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Method of utilizing human umbilical cord mesenchymal stem cells as feed layer to culture human induced pluripotent stem cells

A technology of pluripotent stem cells and pluripotent stem cells, which is applied in the direction of cells modified by introducing foreign genetic material, can solve the problems of limiting the clinical application of human iPSCs, the culture generation is only 14 generations, and the feeder-free medium is expensive. The effect of clinical application prospects

Active Publication Date: 2014-02-19
SICHUAN NEO LIFE STEM CELL BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, using these cells as a feeder layer will inevitably introduce animal-derived components and unknown pathogens, thus limiting the clinical application of human iPSCs
These immunogenic contaminations are difficult to remove from the co-culture system, and the current feeder-free medium for human iPSCs is relatively expensive, so it is particularly urgent and necessary to find human-derived feeder cells suitable for culturing human iPSCs
[0006] In 2010, Christian et al. used human skin fibroblasts as a feeder layer to culture human iPSCs, which can maintain the growth of iPSCs, but the maintenance time is short, and the number of culture generations is only 7 generations; Mesenchymal stem cells are used as a feeder layer to culture human iPSCs, which can maintain the growth of iPSCs, but the maintenance time is still short, and the culture passage is only 14 passages. Moreover, the feeder layer is obtained by culture medium containing fetal bovine serum, containing heterologous proteins, and used There are still potential risks in clinical application

Method used

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  • Method of utilizing human umbilical cord mesenchymal stem cells as feed layer to culture human induced pluripotent stem cells
  • Method of utilizing human umbilical cord mesenchymal stem cells as feed layer to culture human induced pluripotent stem cells
  • Method of utilizing human umbilical cord mesenchymal stem cells as feed layer to culture human induced pluripotent stem cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Isolation, cultivation and identification of human umbilical cord mesenchymal stem cells

[0046] 1. Test method

[0047] 1.1 Isolation and culture method

[0048] Human umbilical cord tissue with a length of about 10 cm was aseptically obtained from the delivered fetus, and the umbilical cord was cut longitudinally in an aseptic manner, and the umbilical artery and umbilical vein were removed, and then washed three times with PBS. Mechanically cut the remaining tissue to about 0.2cm 3 Size, use the tissue block attachment method to attach the tissue block to a 10cm cell culture dish, add the mesenchymal stem cell serum-free medium (Lonza) containing 1% double antibody, and place it at 37°C in 5% CO 2 The cells were cultured in an incubator, and the medium was changed every 3 days. After the cells reached 90% confluence, they were digested with 0.05% trypsin, and subcultured into P1 cells at a ratio of 1:4, and then continued to be subcultured at a ratio of...

Embodiment 2

[0055] The preparation of embodiment 2 feeder layers

[0056] 1. Screening of mitomycin C concentration

[0057] Experimental method: Take the human umbilical cord mesenchymal stem cells (hUCMSC) isolated in Example 1, inoculate them into a 6cm cell culture dish after subculture, and culture them with mesenchymal stem cell serum-free medium (Lonza), and use 5 μg / ml , 10 μg / ml and 15 μg / ml mitomycin C for 3 hours, the group without mitomycin C was used as the blank control group, and the experimental group was washed with PBS for 3 times after treatment, and replaced with serum-free medium for mesenchymal stem cells (Lonza) at 37°C 5% CO 2 cultured in a cell culture incubator. Trypsinization was performed on the 0th day, the 1st day, the 2nd day and the 3rd day after treatment in each group, counted by trypan blue staining, and three replicate wells were set up.

[0058] Experimental results: The cells in the blank control group proliferated normally, while the proliferation...

Embodiment 3

[0063] The preparation of embodiment 3 feeder layer

[0064] Experimental method: the third to seventh generation human umbilical cord mesenchymal stem cells (hUCMSC) isolated in Example 1 were used as feeder layer. Human umbilical cord mesenchymal stem cells were digested with 0.05% trypsin, stained with trypan blue and counted at 0.7×10 4 cells / cm 2 The density was seeded on a 10cm culture dish, cultured in serum-free medium (Life), and the confluence of the cells was about 40% the next day. Add mitomycin C with a final concentration of 10 μg / ml, place it in a cell culture incubator for 2 hours, wash it with PBS three times after treatment, and replace it with a serum-free medium (Life) to obtain human umbilical cord mesenchyme Stem cell feeder layer and used within 24 hours.

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Abstract

The invention discloses a method of utilizing human umbilical cord mesenchymal stem cells as a feed layer to culture human induced pluripotent stem cells. The method comprises the following steps: (1) preparation of the feed layer: taking the human umbilical cord mesenchymal stem cells, inoculating the cells to a culture dish until the cell confluence degree is 40-60%, adding 5-15 ug / ml of mitomycin C, processing the culture for 2-4 h, removing mitomycin C, and obtaining the feed layer; (2) culture: inoculating the human induced pluripotent stem cells to the feed layer which is prepared in the step (1), culturing, and finishing. The method can be used for effectively culturing the human induced pluripotent stem cells for a long time, is free from pollution of heterogonous cells and heterogonous proteins, and has good application prospect.

Description

technical field [0001] The invention relates to a method for cultivating human induced pluripotent stem cells using human umbilical cord mesenchymal stem cells as a feeder layer. Background technique [0002] Stem cells are the most important cells in the human body. Stem cells form different tissues such as muscle, liver, brain and heart. Human embryonic stem cells come from human embryos and can differentiate into various tissue cells of the human body, known as pluripotent stem cells. Studies have shown that human embryonic stem cells can repair damaged organs and treat hearts damaged by myocardial infarction or heart disease. The further application of human embryonic stem cells will open up new avenues for transplantation therapy and treatment of muscular dystrophy, Parkinson's and other intractable diseases. However, since human embryonic stem cells are derived from human embryos, it is unethical to use human embryos to study stem cells. [0003] In 2007, Shinya Yam...

Claims

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Application Information

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IPC IPC(8): C12N5/10
Inventor 邹庆马峰陈强伍明俊钟立武
Owner SICHUAN NEO LIFE STEM CELL BIOTECH
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