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Umbilical cord mesenchymal stem cell primary isolated culture method capable of increasing yield

A technology for improving the yield of stem cells, applied in the field of stem cells, can solve the problems of weak proliferation ability, unstable differentiation ability, long cell growth cycle, etc., and achieve the effect of increasing the amount of acquisition and easy industrialization operation.

Pending Publication Date: 2020-12-25
山东佰傲干细胞生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the primary isolation of umbilical cord mesenchymal stem cells is mostly carried out by tissue method, and there are problems such as immune rejection in the cultivation of human umbilical cord mesenchymal stem cells in the existing serum-containing medium, which cannot meet the needs of future clinical practice; the use of serum-free passage medium Cultivating human umbilical cord mesenchymal stem cells has problems such as long cell growth cycle, poor stability, weak proliferation ability, and unstable differentiation ability. The use of serum-free medium containing various additive factors is expensive and difficult to achieve industrialization requirements.

Method used

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  • Umbilical cord mesenchymal stem cell primary isolated culture method capable of increasing yield
  • Umbilical cord mesenchymal stem cell primary isolated culture method capable of increasing yield
  • Umbilical cord mesenchymal stem cell primary isolated culture method capable of increasing yield

Examples

Experimental program
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Effect test

Embodiment 1

[0034] This embodiment provides a method for primary isolation and culture of umbilical cord mesenchymal stem cells that can increase yield, comprising the following steps:

[0035] (1) Take the umbilical cord tissue, put it into a petri dish with 75% alcohol for disinfection, wash it with normal saline for 3-5 times; cut it off 1cm inside the two ends of the umbilical cord and discard it, and evenly divide the umbilical cord tissue into 2cm~3cm pieces For small sections, wash 3-5 times with normal saline to remove residual blood; cut the umbilical cord along the umbilical vein, remove the umbilical vein and umbilical artery, peel off the Wharton glue, wash 2-3 times, and cut it into pieces with a size of 1mm×1mm×1mm Tissue pieces; in some embodiments, shredded to 1.5mm x 1.5mm x 1.5mm or 2mm x 2m x 2mm sized tissue pieces;

[0036] (2) Evenly inoculate the shredded tissue pieces in T175 culture flasks, add culture medium, and culture in an incubator at a temperature of 37°C a...

Embodiment 2

[0050] This example is a comparative example of Example 1. The difference between this example and Example 1 is that the concentration of carbon dioxide in step (2) of Example 1 is adjusted to 5%.

[0051] Count the amount of cell acquisition and cell activity, and the results are shown in Table 1. Table 1 is a comparison chart of the number and cell activity of primary cells obtained by different methods; wherein the control group adopts the culture method of Example 2; The technical scheme in Example 1 of the present invention; Experimental Group 2 is to adjust the carbon dioxide content of step (2) in Example 1 of the present invention to 1.5%, and Experimental Group 3 is to adjust the carbon dioxide content of step (2) in Example 1 of the present invention The content was adjusted to 2%. It can be seen that the acquisition amount and cell activity of mesenchymal stem cells obtained by the experimental group 1-3 were significantly higher than the cell acquisition rate of the...

Embodiment 3

[0054] Example 3 Stability identification of high-yield culture of umbilical cord mesenchymal stem cells

[0055] The ratio of positive CD73, CD90 and CD105 surface markers and the negative ratio of CD14, CD19, CD34, CD45 and HLA-DR in the P5, P10 and P20 generations of human umbilical cord mesenchymal stem cells cultured by the method described in Example 1 , P5, P10, P20 human umbilical cord mesenchymal stem cells were digested with 0.25mg / ml trypsin and counted, diluted to 1×10 5 -1×10 6 per L, divided into six groups, each group was added with a diluted monoclonal antibody, the monoclonal antibody is CD73 monoclonal antibody, CD90 monoclonal antibody, CD105 monoclonal antibody, CD14 monoclonal antibody, CD19 monoclonal antibody Six kinds of cloned antibody, CD34 monoclonal antibody, CD45 monoclonal antibody, and HLA-DR monoclonal antibody. The diluent is 1% fetal calf serum in phosphate buffer. The ratio of the above monoclonal antibody to the diluent is 1:1000, at room t...

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Abstract

The invention provides an isolated culture method capable of increasing the yield of umbilical cord mesenchymal stem cells. According to the isolated culture method, the number of obtained umbilical cord mesenchymal stem cells is increased mainly through the volume of tissue blocks, the change of the oxygen concentration in a primary culture process, the addition of an antioxidant with high cost performance and the like. According to the method, the obtaining amount of the umbilical cord mesenchymal stem cells is increased through an economical and practical primary separation mode, and the method is simple, economical and easy for industrial operation.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and in particular relates to a method for primary isolation and culture of umbilical cord mesenchymal stem cells that can increase yield. Background technique [0002] Mesenchymal stem cells (Mesenchymal stem cells, MSC) are a type of stem cells with self-renewal, proliferation and multi-directional differentiation potential. , no allogeneic rejection, avoid ethical controversy, stimulate tissue regeneration, regulate immune function and many other advantages. [0003] At present, umbilical cord mesenchymal stem cells (UC-MSC, Umbiliacl cord mesenchymal stem cells) have good therapeutic effects in cartilage regeneration, cerebral ischemic diseases, diabetes, rheumatoid arthritis and other animal models and clinical studies, showing Broad application prospects. At home and abroad, there is no unified standard for the isolation, culture and expansion of umbilical cord mesenchymal stem cells. ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2509/10C12N2509/00C12N2501/30C12N2500/38
Inventor 程蕊苹
Owner 山东佰傲干细胞生物技术有限公司
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