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Isolation method and kit for high-purity umbilical cord blood stem cells

A technology of umbilical cord blood stem cells and separation methods, applied in cell dissociation methods, animal cells, vertebrate cells, etc., can solve the problems of danger, no attention to the purity of effective stem cells, etc., and achieve the effect of high purity

Active Publication Date: 2019-12-03
BEI ZHENG STEM CELLS BIOLOGICAL TECH CO LTD BEIJING
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the purpose of these technologies is to improve the recovery rate and viability of the obtained product, and the purity of the effective stem cells in the obtained product is not paid attention to.
[0004] The presence of impurities can be extremely detrimental and even dangerous for stem cells used in therapy, especially directly for injection

Method used

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  • Isolation method and kit for high-purity umbilical cord blood stem cells
  • Isolation method and kit for high-purity umbilical cord blood stem cells
  • Isolation method and kit for high-purity umbilical cord blood stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] First, 50 ml of umbilical cord blood sample containing sodium citrate was mixed with 50 ml of 0.9% by weight sodium chloride aqueous solution to form a mixed solution.

[0046] Then, 6% by weight of hydroxyethyl starch aqueous solution was added to the above mixed solution and left to settle for 1 hour to obtain the supernatant cell liquid containing stem cells, which was centrifuged at 2500 rpm for 10 minutes to obtain Cell pellet. The cell pellet was spread on an aqueous solution with a density of 1.075 g / ml prepared by polysucrose and diatrizoate for centrifugation, wherein the mass of polysucrose and diatrizoate was 0.8:1. Then centrifuge at 2000 rpm for 20 minutes to obtain the middle cloudy stem cell layer. Take the middle stem cell layer, spread it on the polysucrose aqueous solution with a density of 1.092g / ml in another test tube, and centrifuge at a centrifugal speed of 1700 rpm for 35 minutes to obtain two upper and lower cell bands, and take the lower cell ...

Embodiment 2

[0048] Effective cells were prepared in the same manner as in Example 1, except that 1% adenine was added to the cell purification solution. The conditions of the cells included are shown in Table 1.

[0049] As a control, 50ml of umbilical cord blood containing sodium citrate anticoagulant was separated according to the method provided in Example 1 of Chinese Patent Publication CN101638637A to obtain a precipitate containing stem cells, the cells contained in which are listed in Table 1 Show.

[0050] Table 1: Comparison of the stem cells isolated by the method of the present invention and the prior art

[0051]

[0052] Note:

[0053] CD detection by flow cytometry 34 Number of cells, number of total cells after isolation, and total number of anucleated cells or cells with damaged nuclei;

[0054] Recovery = CD after separation 34 Cell number / CD before isolation 34 Cell number × 100%;

[0055] Purity = CD after separation 34 Cell number / total cell number after iso...

Embodiment 3

[0058] First, 30ml of cord blood sample containing sodium citrate was mixed with 35ml of PBS solution to form a mixture.

[0059] Then add 6% by weight of hydroxyethyl starch aqueous solution to the above mixed solution and let it settle for 1 hour to obtain the supernatant cell liquid containing stem cells, which is centrifuged at 2600 rpm for 10 minutes to obtain Cell pellet. The cell pellet was spread on an aqueous solution with a density of 1.075 g / ml prepared by polysucrose and centrifuged, wherein the mass of polysucrose and diatrizoate was 0.85:1. Then centrifuge at 2000 rpm for 20 minutes to obtain the middle cloudy stem cell layer. Take the middle stem cell layer, spread it on the polysucrose aqueous solution with a density of 1.092g / ml in another test tube, and centrifuge at a centrifugal speed of 1800 rpm for 32 minutes to obtain two upper and lower cell bands, and separate the lower cell band. That is, the effective cells described in the present invention. The ...

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Abstract

The invention relates to a method and a kit for separating high-purity umbilical cord blood stem cells. The method comprises steps as follows: dilution, precipitation, layering and purification, wherein a cell diluent in the dilution step is a PBS (poly butylenes succinate) solution or 0.8%-1.0% of a sodium chloride aqueous solution by weight, a cell precipitant in the precipitation step is 4%-10% of a hydroxyethyl starch aqueous solution by weight, a cell layering solution in the layering step is an aqueous solution prepared from polysucrose and / or meglumine diatrizoate and having the density of 1.074-1.076 g / ml, and a cell purifying solution in the purification step is an aqueous solution prepared from polysucrose and having the density of 1.090-1.095 g / ml. With the adoption of the method and the kit, higher-purity effective stem cells can be obtained, the number of impurity cells is lower, and great significance in the clinical application is realized.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method and a kit for separating high-purity umbilical cord blood stem cells. Background technique [0002] Cord blood is the blood that remains in the placenta and umbilical cord after the baby is delivered and the umbilical cord is ligated and severed. Research in the past ten years has found that umbilical cord blood contains hematopoietic stem cells that can rebuild human hematopoietic and immune systems, and can be used for hematopoietic stem cell transplantation to treat more than 80 diseases. Therefore, cord blood has become an important source of hematopoietic stem cells, especially unrelated hematopoietic stem cells. Therefore, umbilical cord blood is a very important human biological resource. [0003] One of the most important components of cord blood are cord blood stem cells. Therefore, isolating or extracting these stem cells from umbilical cord blood is a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2509/00
Inventor 唐明淇赵涌
Owner BEI ZHENG STEM CELLS BIOLOGICAL TECH CO LTD BEIJING
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