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Separation and culture method of umbilical cord blood derived mesenchymal stem cells

A technology for the isolation and cultivation of stem cells, applied in the biological field, can solve problems such as unstable sources, fluctuations in serum batch differences, and unclear components, and achieve the effects of low cost, easy operation, and simple process

Pending Publication Date: 2020-05-19
陕西九州细胞基因工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, due to the low content of mesenchymal stem cells contained in human umbilical cord blood, the requirements for its isolation and culture are relatively high. At present, there are few reports on the isolation of human umbilical cord blood mesenchymal stem cells in China; In the culture method of umbilical cord blood mesenchymal stem cells in vitro, a certain concentration of fetal bovine serum is added to the medium, mainly to provide nutrients such as hormones, growth factors, and transport proteins for the growth and proliferation of mesenchymal stem cells, but this There are many shortcomings in the method: such as fluctuations in differences between batches of serum, unclear ingredients, unstable sources and other reasons, a lot of verification work is required before use, which will complicate production and scientific research

Method used

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  • Separation and culture method of umbilical cord blood derived mesenchymal stem cells
  • Separation and culture method of umbilical cord blood derived mesenchymal stem cells
  • Separation and culture method of umbilical cord blood derived mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0033] This embodiment provides a method for isolating and culturing umbilical cord blood mesenchymal stem cells, which includes the following steps:

[0034] (1) Collect human umbilical cord blood, and place the umbilical cord blood in a 50mL centrifuge tube within 2 hours. After balancing, perform centrifugation at 2000rpm for 10 minutes, take the upper layer, collect plasma, and the lower layer is cells.

[0035] (2) Add the plasma collected from the upper layer to a new centrifuge tube, place it in a water bath at 56°C for 30 minutes, and then centrifuge it at a speed of 4000 rpm for 5 minutes to collect the upper layer to obtain autologous serum. Store in a refrigerator at 4°C for subsequent coating of culture dishes and termination of digestion during passage; standby

[0036] (3) Add physiological saline to the lower layer of cells obtained by centrifugation in the above step 1 to the original volume, and after mixing evenly, take 25 mL and slowly add it to a centrifuge...

Embodiment 2

[0041] This embodiment provides a method for isolating and culturing umbilical cord blood mesenchymal stem cells, which includes the following steps:

[0042] (1) Collect human umbilical cord blood, and place the umbilical cord blood in a 50mL centrifuge tube within 2 hours. After balancing, perform centrifugation at 2000rpm for 10 minutes, take the upper layer to obtain plasma, and the lower layer to obtain cells.

[0043] (2) Add the plasma collected above to a new centrifuge tube, place it in a water bath at 56°C for 30 minutes, and then centrifuge it at 4000 rpm for 5 minutes. Collect the upper layer to obtain autologous serum and store it. Store in a refrigerator at 4°C for subsequent coating of culture dishes and termination of digestion during passage.

[0044] (3) Add physiological saline to the lower layer of cells obtained by centrifugation in the above step 1 to the original volume, and after mixing evenly, take 25 mL and slowly add it to a centrifuge tube containin...

Embodiment 3

[0049] This embodiment provides a method for isolating and culturing umbilical cord blood mesenchymal stem cells, which includes the following steps:

[0050] (1) Collect human umbilical cord blood, and place the umbilical cord blood in a 50mL centrifuge tube within 2 hours. After balancing, perform centrifugation at 2000rpm for 10 minutes, take the upper layer to obtain plasma, and the lower layer to obtain cells.

[0051] (2) Add the plasma collected above to a new centrifuge tube, place it in a water bath at 56°C for 30 minutes, and then centrifuge it at 4000 rpm for 5 minutes. Collect the upper layer to obtain autologous serum and store it. Store in a refrigerator at 4°C for subsequent coating of culture dishes and termination of digestion during passage.

[0052] (3) Add physiological saline to the lower layer of cells obtained in the above step 1 to the original volume, mix well, take 25mL and slowly add it to a centrifuge tube containing 20mL lymphocyte separation mediu...

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Abstract

The invention discloses a separation and culture method of umbilical cord blood derived mesenchymal stem cells, and belongs to the technical field of biology. The separation and culture method comprises the following steps of performing centrifuging treatment on umbilical cord blood, taking an upper layer to obtain plasma, and taking a lower layer to obtain cells; performing inactivation treatmenton the plasma, then performing centrifuging treatment, and taking supernatant so as to obtain autoserum; mixing the lower-layer cells with normal saline to obtain a mixture, then placing the mixturein lymphocyte separation liquid, and performing centrifuging treatment so as to obtain umbilical cord blood mononuclear cells; and re-suspending the umbilical cord blood mononuclear cells with serum-free culture mediums, placing the re-suspended umbilical cord blood mononuclear cells in an autoserum coating culture dish, performing primary culture to obtain primary mesenchymal stem cells, adding cell dissociation liquid into the primary mesenchymal stem cells, performing cell dissociation treatment, and then performing subculturing. According to the method, penicllin-streptomycin for cell culture is not added in a separation process and a culture process, so that toxicity is not generated on the cells. In addition, according to the method, the autoserum coating culture dish for centrifugalseparation is adopted, so that animal derived viruses cannot be introduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating and culturing umbilical cord blood mesenchymal stem cells. Background technique [0002] Human umbilical cord blood-derived MSCs (Human umbilical cord blood-derived MSCs, hUCB-MSCs) are a kind of adult stem cells in human umbilical cord blood, which have strong self-renewal ability and the potential to differentiate into multiple germ layers. Under certain conditions, hUCB-MSCs can differentiate into bone cells, adipocytes, chondrocytes, etc., and can secrete a variety of cell activity factors, which have the functions of immune regulation, inhibition of inflammation, and reduction of apoptosis. In addition, hUCB-MSCs also have the advantages of low immunogenicity, wide range of sources, and less ethical controversy. Compared with bone marrow-derived and adipose-derived mesenchymal stem cells, human cord blood-derived mesenchymal stem cells have a stronger pr...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2500/90C12N2509/10
Inventor 何婷婷聂苏秦肖轩戴博朱艳丽
Owner 陕西九州细胞基因工程有限公司
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