Culture medium for induced pluripotent stem cells and application of culture medium

A technology of pluripotent stem cells and pluripotent stem cells, applied in the field of pluripotent stem cell culture medium, can solve problems such as gaps

Active Publication Date: 2014-12-10
INST OF ZOOLOGY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The early iPSC induction efficiency is only 0.01-0.5%, which is far from the reprogramming efficiency based on somatic cell nucle

Method used

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  • Culture medium for induced pluripotent stem cells and application of culture medium
  • Culture medium for induced pluripotent stem cells and application of culture medium
  • Culture medium for induced pluripotent stem cells and application of culture medium

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0055] Example 1 Preparation of culture medium

[0056] KOSR medium is a conventional medium for culturing stem cells. The KOSR medium in this example was purchased from Invitrogen. The formula is shown in Table 1

[0057] Table 1 Formula of KOSR medium

[0058]

[0059] Dissolve ginsenoside monomer Rb1, ginsenoside monomer Rb3, and ginsenoside monomer Rd in a trace amount of DMSO to make a certain concentrated stock solution, and then add a corresponding volume of concentrated stock solution to the above KOSR medium as needed to make ginseng The concentration gradients of saponin monomer Rb1, ginsenoside monomer Rb3, and ginsenoside monomer Rd are 1 μM, 5 μM, 25 μM, 50 μM, 75 μM, 100 μM. The DMSO control group is the same volume of DMSO as the ginsenoside monomer concentrated stock solution added to the above-mentioned KOSR medium; the positive control group is the KOSR medium containing 50μg / ml Vc.

Example Embodiment

[0060] Example 2 Induction and culture of mouse iPS cells

[0061] The inventors of the present application respectively prepared a medium containing ginsenoside monomer Rb1, ginsenoside monomer Rb3, and ginsenoside monomer Rd. The concentrations of ginsenoside in the culture medium were 1 μM, 5 μM, 25 μM, 50 μM, 75 μM, respectively. , 100μM.

[0062] In the following experimental procedures, except for the above-mentioned medium, the rest are the same.

[0063] The negative control is KOSR medium containing an equal volume of DMSO; the positive control is KOSR medium containing 50ug / ml Vc.

[0064] The medium prepared in Example 1 was used to induce mouse ips cells, and MEF medium (10% DMEM) was also used. The formula is shown in Table 1:

[0065] Table 1 MEF culture medium

[0066]

[0067] The method includes the following steps:

[0068] First, the pluripotency-related genes (Klf4, Sox2, Nanoge, and Oct4) are introduced into the donor cell by the following method:

[0069] 1) Press 1...

Example Embodiment

[0081] Example 3 The effect of adding ginsenoside monomer culture solution on iPS induction

[0082] 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 mice through tetraploid complementation. Nature, 2009.461(7260): p.86-90. The method described in observing and identifying the state of cells during induction includes:

[0083] 2.1 alkaline phosphatase (AP) staining method

[0084] Select the cells induced by the final concentration of 25μM ginsenoside monomer culture solution, fix the cells in the culture plate on the 10th day, after AP staining, count the number of colored clones to determine the number of clones.

[0085] For specific embodiments, refer to the instructions of the BCIP / NBT alkaline phosphatase staining kit.

[0086] The result is figure 1 As shown, it shows the statistical result...

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Abstract

The invention provides a culture medium for culturing induced pluripotent stem cells. The culture medium contains a ginsenoside monomer Rb1, a ginsenoside monomer Rb3 and/or a ginsenoside monomer Rd. The invention also provides a method for inducing pluripotent stem cells. The method comprises the following steps: (1) introducing pluripotent-related genes into donor cells; (2) preparing the donor cells in which the pluripotent-related genes are introduced into single-cell suspension, and culturing the donor cells (in which the pluripotent-related genes are introduced in the step (1)) by using the culture medium provided by the invention; and (3) identifying cloning of the pluripotent stem cells. In consideration of the application bottleneck of ips to clinical test at present, the ginsenoside monomer Rb1, the ginsenoside monomer Rb3 and the ginsenoside monomer Rd are discovered, the ips induction efficiency can be obviously improved, cell senescence can be obviously delayed, cell proliferation can be promoted, and far-reaching influence is generated in the entire field of stem cell research and new drug research and development.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the use of ginsenosides, in particular to the use of a pluripotent stem cell culture medium containing ginsenoside monomers in improving cell reprogramming efficiency, delaying cell aging and promoting cell proliferation. technical background [0002] Chinese herbal medicine provides a broad source of medicine and plays an important role in traditional medicine. Some traditional Chinese medicines have significant curative effects on brain development and immunity, but the specific molecular mechanisms are still unclear, including targets, signaling pathways, etc. In traditional Chinese medicine, ginseng can enhance memory, improve eyesight and calm the mind. Analysis found that ginseng contains a specific active substance - ginsenoside (ginsenoside, GS). Ginsenosides have similar basic chemical structures and can be roughly classified into dammarane type and oleanane type. Due to the...

Claims

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Application Information

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IPC IPC(8): C12N5/0735A61K31/704A61P39/06A61P35/00A23L1/30A23L33/10
Inventor 周琪王秀杰顾奇郝捷韩伟方刘鑫王磊王柳
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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