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Separation preparation method of umbilical cord blood stem cells

A technology for umbilical cord blood stem cells and umbilical cord blood, which is applied in the separation field with high stem cell recovery rate and high activity, can solve the problems of unclean removal of red blood cells, small number of target cells, and small number of stem cells collected, so as to avoid the decline of cell activity and avoid The effect of low separation purity and improved final purity

Pending Publication Date: 2020-12-04
中康再生医学科技(海南)有限公司
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AI Technical Summary

Problems solved by technology

The above several separation and extraction methods can realize the separation of umbilical cord blood stem cells, but in the actual application process, there are mainly the following defects: (1) The health status of stem cells cannot be guaranteed, and the umbilical cord blood stem cells obtained by separation are of low quality, which affects the efficacy of cell therapy (2) Most of them are only suitable for small batches of umbilical cord blood separation, which cannot meet the large demand of clinical treatment; (3) When separating umbilical cord blood stem cells in large quantities, the workload is heavy and it is easy to cause cross-contamination; (3) The separation effect is poor, and stem cells The number of collections is small and the purity is low, that is, the collected cells are relatively miscellaneous, and the number of target cells that can be used for treatment is small, and for cord blood, using this method to remove red blood cells is not clean, and the final clinical application will cause patients Rejection reaction occurs; (4) The cost of the equipment used separately is high, resulting in higher treatment costs and increased treatment costs for patients, which is not conducive to the promotion and popularization of the project

Method used

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  • Separation preparation method of umbilical cord blood stem cells

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Experimental program
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Effect test

Embodiment 1

[0024] Embodiment 1: A method for separating and preparing umbilical cord blood stem cells, comprising the following steps:

[0025] S1, collection of umbilical cord blood: after the placenta of the parturient falls off, use a blood collection bag containing sodium citrate or heparin anticoagulant, insert the needle on the blood collection bag into the umbilical vein, and gently shake the blood collection bag while collecting, so that The anticoagulant is fully mixed with the umbilical cord blood to obtain the pretreated sample of umbilical cord blood;

[0026] S2, pour the umbilical cord blood pretreatment sample into a large-capacity sterile culture bottle, add a diluent containing cerebroprotein hydrolyzate and normal saline into the culture bottle at a volume ratio of 1:1, cover the bottle cap, and shake well to obtain Dilute the cell solution, the mass fraction of the cerebroprotein hydrolyzate is 5%, and the mass fraction of sodium chloride in the normal saline is 10%; ...

Embodiment 2

[0030] Embodiment 2: A method for separating and preparing umbilical cord blood stem cells, the only difference from Example 1 is that in the diluent described in step S2, the mass fraction of cerebroprotein hydrolyzate is different from the mass fraction of sodium chloride in normal saline, as follows As shown in the table:

[0031] Table 1

[0032]

Embodiment 3

[0033] Embodiment 3: The cell precipitation agent in Embodiment 1 and Embodiment 2 can also be: a methylcellulose solution with a mass fraction of 10-20%, and this solution uses 8% physiological saline as a solvent.

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Abstract

The present invention relates to a separation preparation method of umbilical cord blood stem cells. The method specifically comprises the following steps: S1, collecting umbilical cord blood; S2, adding a diluent containing a cerebroprotein hydrolysate and normal saline into an umbilical cord blood pretreatment sample according to a volume ratio of 1:1 to obtain a diluted cell solution; S3, adding an erythrocyte precipitator into the diluent, transferring an upper-layer cell solution into a centrifugal tube for sub-packaging, and performing gradient centrifugal separation in three steps; S4,transferring middle-layer umbilical cord blood stem cells into another centrifugal tube; S5, adding normal saline into the centrifuge tube for collecting the umbilical cord blood stem cells for dilution, carrying out centrifugal separation after gently and uniformly mixing, setting conditions of the centrifugal separation to be 500 gx5 min, and washing a precipitation layer with the normal salineafter centrifuging; wherein the normal saline contains the cerebroprotein hydrolysate with a concentration of 1-3%, and finally collecting the umbilical cord blood stem cells at a bottom part of the centrifugal tube. The separation preparation method of the umbilical cord blood stem cells can separate and purify the umbilical cord blood stem cells on a large scale and has high recovery rate and activity of the stem cells.

Description

technical field [0001] The invention relates to a method for separating and preparing umbilical cord blood stem cells, in particular to a separation method which can separate and purify umbilical cord blood stem cells in large quantities, and has high stem cell recovery rate and high activity. Background technique [0002] Umbilical cord blood hematopoietic stem cells are the origin cells of all blood cells and immune cells, with the potential of self-renewal, multi-directional differentiation and self-replication, and can be used for leukemia, immune system diseases, innate immune diseases, autoimmune diseases, solid tumors, ovarian cancer , neuroblastoma and other diseases. [0003] At present, the technology of separating umbilical cord blood stem cells from umbilical cord blood is called: umbilical cord blood separation technology. This technology is mainly used to remove red blood cells, platelets and part of the plasma in cord blood, collect nucleated cells rich in co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2509/00
Inventor 李伟王熙凯赵庆义
Owner 中康再生医学科技(海南)有限公司
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