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Culture method of human umbilical cord blood mesenchymal stem cells

A technology of mesenchymal stem cells and human umbilical cord blood, applied in the field of cell biology, can solve the inevitable problems of mesenchymal stem cell special medium use, separation, high cost of culture, unsuitable for large-scale production, etc., to avoid special medium The use, good adipogenic differentiation ability, the effect of promoting growth

Active Publication Date: 2020-06-05
XINXIANG MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, Mesenscult TM The price of the medium is expensive, resulting in a significant increase in the cost of separation and culture, especially not suitable for large-scale production
[0006] The Chinese invention patent with the notification number CN102559590B discloses a method for sequentially culturing human umbilical cord blood mesenchymal stem cells using two culture media. DMEM / F12 medium was cultured from the primary culture to the second passage, and from the third passage to the Oricell human umbilical cord mesenchymal stem cell medium for subculture to the eighth passage. The cultured mesenchymal stem cells can maintain hUCB - MSCs have good biological characteristics, but the above-mentioned method of sequentially culturing human umbilical cord blood mesenchymal stem cells using two media still inevitably uses a special medium for mesenchymal stem cells, and there is still a problem of high cost, which is not conducive to obtaining A large number of blood mesenchymal stem cells to meet the demand for a large amount of use in the treatment of related diseases

Method used

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  • Culture method of human umbilical cord blood mesenchymal stem cells
  • Culture method of human umbilical cord blood mesenchymal stem cells
  • Culture method of human umbilical cord blood mesenchymal stem cells

Examples

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Embodiment 1

[0051] The method for culturing human umbilical cord blood mesenchymal stem cells of the present embodiment comprises the following steps:

[0052] (1) Separation of mononuclear cells:

[0053] 1) The umbilical cord blood of premature infants was extracted under sterile conditions, and anticoagulated with heparin;

[0054] 2) Use D-Hanks balanced salt solution to dilute heparin to anticoagulate the umbilical cord blood in equal proportions;

[0055] 3) superimposing the diluted heparin anticoagulated umbilical cord blood on the Ficoll lymphocyte separation medium to obtain a mixed solution, the height ratio of the diluted heparin anticoagulated umbilical cord blood to the lymphocyte separation medium is 2:1;

[0056] 4) Centrifuge the mixture obtained in step 3) at 2000r / min for 20min, carefully absorb the white cloud layer at the interface,

[0057] Centrifuge with PBS balanced salt solution at 1000r / min to wash the precipitate twice.

[0058] (2) Culture of umbilical cord...

Embodiment 2

[0067] The method for culturing human umbilical cord blood mesenchymal stem cells of the present embodiment comprises the following steps:

[0068] (1) The isolation method of mononuclear cells is the same as in Example 1.

[0069] (2) Culture of umbilical cord blood mesenchymal stem cells in premature infants:

[0070] 1) adding the mononuclear cells isolated in step (1) to the first culture medium to make a single cell suspension, and counting the cells;

[0071] 2) According to the cell density 1×10 7 Individual / mL, the mononuclear cells in step 1) were inoculated in gelatin-coated T25 cell culture flasks;

[0072] 3) Place the cell culture flask at 37°C with a volume fraction of 5% CO 2 Cultivate in a saturated humidity incubator, change the medium after 2 days, change the medium for the first time after 5-7 days, and change the medium every 3-4 days, and observe the growth and morphological characteristics of the primary cells every day under an inverted phase-contrast...

Embodiment 3

[0078] The method for culturing human umbilical cord blood mesenchymal stem cells of the present embodiment comprises the following steps:

[0079] (1) The isolation method of mononuclear cells is the same as in Example 1.

[0080] (2) Culture of umbilical cord blood mesenchymal stem cells in premature infants:

[0081] 1) adding the mononuclear cells isolated in step (1) to the first culture medium to make a single cell suspension, and counting the cells;

[0082] 2) According to the cell density 2×10 7Individual / mL, the mononuclear cells in step 1) were inoculated in gelatin-coated T25 cell culture flasks;

[0083] 3) Place the cell culture flask at 37°C with a volume fraction of 5% CO 2 Cultivate in a saturated humidity incubator, change the medium after 2 days, change the medium for the first time after 5-7 days, and change the medium every 3-4 days, and observe the growth and morphological characteristics of the primary cells every day under an inverted phase-contrast ...

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Abstract

The present invention belongs to the field of cell biology and particularly relates to a culture method of human umbilical cord blood mesenchymal stem cells. The method comprises the following steps:umbilical cord blood mononuclear cells are cultured with a first culture medium until cell growth state is stable, the umbilical cord blood mononuclear cells are subsequently cultured with a second culture medium, and the first culture medium is mainly composed of a basic culture medium, fetal bovine serum, antibiotics and a mesenchymal stem cell growth additive, wherein a volume ratio of the fetal bovine serum accounts for 8-12% and a volume ratio of the mesenchymal stem cell growth additive accounts for 0.1-0.2%; the second culture medium is mainly composed of a basic medium, fetal bovine serum and antibiotics, wherein a volume ratio of the fetal bovine serum is 8-12%; and the basic culture medium is a DMEM / F12 culture medium. The culture method of the human umbilical cord blood mesenchymal stem cells is simple in steps, relatively low in economic cost, high in culture success rate, and suitable for obtaining a large number of the mesenchymal stem cells to meet a demand for a relatively large use amount in treatments of related diseases.

Description

technical field [0001] The invention belongs to the field of cell biology, and in particular relates to a method for culturing human umbilical cord blood mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (Mesenchymal stem cells, MSCs) have the potential of self-renewal and multi-lineage differentiation, and have become a very valuable cell source in the field of gene therapy. MSCs were initially isolated from bone marrow and are the most widely used, and MSCs can also be isolated from other tissues and organs. At present, MSCs from different tissue sources, such as umbilical cord blood, menstrual blood, and adipose tissue, have been used in the treatment of clinical diseases. Among them, human umbilical cord blood is a good source of cells. It is not from the mother's blood, but belongs to the fetus' own blood, which is not confused with the mother's blood. However, umbilical cord blood is usually discarded as "garbage" during childbirth, and its...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2500/84C12N2509/00
Inventor 张俊河柴树洁张继红高建辉单琳琳杨雯雯杨献军
Owner XINXIANG MEDICAL UNIV
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