Culture system for inducing pluripotent stem cells

A technology of pluripotent stem cells and pluripotent stem cells, applied in the field of culture system of induced pluripotent stem cells, can solve the problems of differences in molecular expression markers, unsatisfactory effects, low induction efficiency, etc., and achieve good gene modification and low cost , the effect of improving the induction efficiency

Active Publication Date: 2015-09-23
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2009, the first porcine iPS cell line was established, and then it was replicated in multiple laboratories, but the molecular expression markers were somewhat different, especially for SSEA1 and SSEA4, but it was disappointing that the expression of foreign genes was to maintain pluripotency Necessary for status, although chimera experiments have been reported to be possible, so far have not been replicated by other laboratories
However, the effect is not ideal. In view of the above problems, the present invention explores and optimizes the induced culture system first, and can obtain porcine iPS with high efficiency, and these porcine iPS are more inclined to mouse-like clones, and can be passaged by single cells. Higher developmental potential and better transgenic efficiency
Since cells from mice, monkeys and humans also suffer from low induction efficiency, there is currently a need for a medium capable of inducing pluripotent stem cells and improving their induction efficiency

Method used

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  • Culture system for inducing pluripotent stem cells
  • Culture system for inducing pluripotent stem cells
  • Culture system for inducing pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1 Preparation of LBX liquid medium

[0096] Prepare LBX liquid medium A-E according to the components and ratios provided in Tables 1-3. The steps of the medium are as follows:

[0097] 1) According to the total volume of preparation, calculate the amount of various components required;

[0098] 2) Dissolve the water-soluble components in the components directly in sterilized deionized water, and stir evenly;

[0099] 3) Dissolve other components in a small amount of DMSO; wherein the amount of DMSO used does not exceed 0.5% of the total volume;

[0100] 4) After mixing the two obtained in step 2) and step 3) uniformly; dilute the volume to the stated total volume; filter it with a 0.22 micron filter.

[0101] Table 1 Composition and ratio of medium A and B,

[0102] Unless otherwise specified, the concentration unit of each component in Table 1 is mg / l

[0103]

[0104]

[0105]

[0106]

[0107] Table 2 Composition and ratio of medium C and D

[0108] Unless otherwise spec...

Embodiment 2

[0116] Example 2 Using the culture medium of the present invention to induce pluripotent stem cells

[0117] Specific implementation steps:

[0118] Prepare porcine fibroblasts (purchased from the Institute of Zoology, Chinese Academy of Sciences) one day in advance, press 2*10 4 / cm 2 Inoculated on 10% DMEM medium.

[0119] One day later, change the medium to 10% DMEM that does not contain sp, and then add the virus packed with the four factors Klf4, Sox2, Nanoge, and Oct4 to the pre-inoculated porcine fibroblasts at the same time, adding 8μg / ml at the same time Polybrene (increase infection efficiency).

[0120] The induction steps of the ips cells are as follows:

[0121] Preliminary preparation: press 2*10 for cells to be induced 4 / cm 2 Inoculated on 10% DMEM medium containing SP.

[0122] Day 0: Change the medium to 10% DMEM without sp. Then add the virus packed with the four factors Klf4, Sox2, Nanoge, and Oct4 to the pre-inoculated cells at the same time, and add 8μg / ml polybre...

Embodiment 3

[0129] Example 3 Ratio of induced pluripotent stem cells using the medium of the present invention and KOSR medium More

[0130] Use the medium E and KOSE medium in Example 1 to induce porcine pluripotent stem cells according to the method in Example 2; and analyze

[0131] Among them, the KOSR medium is a conventional medium for culturing stem cells, the KOSR medium in this embodiment;

[0132] KOSR culture medium is prepared from the following finished products 78%KODMEM+20%KOSR+1%SP+1%L-glu+1%NEAA+0.1%β-ME, the above reagents are purchased from Life Technology Company:

[0133] Wherein, the ratio is the volume percentage, and the product number or other information of each component is shown in Table 4:

[0134] Table 4

[0135]

[0136]

[0137] Using Wang, J., et al., Generation of Induced Pluripotent Stem Cells with High Efficiency from Human Umbilical Cord Blood Mononuclear Cells.Genomics Proteomics Bioinformatics.11(2013):304-311 and Zhao, XY, et al., iPS cells produce viable ...

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Abstract

The invention provides a medium for inducing pluripotent stem cells. The medium is an LBX liquid medium, and the stem cells are porcine stem cells, mouse stem cells, monkey stem cells or human stem cells. The invention also provides a method for inducing the pluripotent stem cells by using the medium. The medium for well improving the induction efficiency of induction pluripotency is invented for the first time. The cost of the medium is low. Induced pluripotent stem cells induced in the invention are more likely to be in a mouse-like state, and single cell passage can be realized, so large-scale amplification and clinic application are benefited.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and relates to a culture system for induced pluripotent stem cells. Specifically, the invention relates to a culture system for induced pluripotent stem cells, a preparation method and application thereof. Background technique [0002] The establishment of mouse embryonic stem cells (ES) and human embryonic stem cells has created a new field of biology and medicine. Embryonic stem cells have the ability to self-renew and differentiate into cells of various germ layers to provide powerful research on individual development and cell differentiation. Tool, and has broad application prospects in the field of regenerative medicine and tissue engineering. It will soon be applied to cell therapy and disease model research. It can provide replacement cells for tissue regeneration for diseases that humans cannot repair themselves (Parkinson, diabetes, etc.) . [0003] The establishment of pig embryonic stem cells w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10
Inventor 周琪顾奇郝捷王柳
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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