Culture medium additive and application thereof

A medium additive, complete medium technology, applied in cell culture active agent, culture process, tissue culture and other directions, can solve the problems of slow reprogramming process, decreased reproducibility, serum instability, etc., to accelerate the iPS process , The effect of improving the efficiency of iPS

Active Publication Date: 2011-11-09
杭州健崃生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of serum culture system has the following disadvantages: 1. The reprogramming process is slow
In addition, other unknown components in the serum are also very likely to delay the reprogramming process
These proven and unproven substances make reprogramming extremely inefficient
2. Batch-to-batch instability in serum
The serum preparation process determines that the composition of each batch of serum is different, which can easily lead to a decrease in the repeatability of the experiment
3. Uncertainty in the composition of serum creates obstacles for the study of reprogramming mechanism and drug screening
[0004] However, the components of the system are still not completely clear. For example, the bovine serum globulin complex (Albumax) contains a variety of unknown lipids, and the impact of these components on reprogramming is still unknown
Second, serum replacement (KOSR) cannot support the growth of pre-reprogramming cells
In addition, the reprogramming efficiency is not high enough even when mixed with serum during iPS induction in mice

Method used

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  • Culture medium additive and application thereof
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Examples

Experimental program
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Effect test

preparation example Construction

[0177] The process of cell experiment and virus preparation is as follows: the cells are planted in 12-well adherent cell culture plate, and the planting density is 20000 cells / well. 6-18 hours after the cells were planted, the cells were infected with the virus containing the mouse reprogramming factor depending on the cell density and state. Infection is carried out in two rounds, the second round of infection is carried out 24 hours after the first round of infection, and the virus liquid is replaced with various culture liquids to be tested 24 hours after the second round of infection. The day of medium change was recorded as day 0 (D0); at different time points after infection, count the number of GFP fluorescent clones in the original well or use flow cytometry to analyze the ratio of GFP fluorescent cells according to the needs of the experiment.

[0178] The preparation process of the virus is as follows: the reprogramming factor plasmid cloned on the PMX vector is tra...

Embodiment 1

[0181] Example 1: Comparison of reprogramming efficiency and component concentration optimization between B3.0 and other culture schemes.

[0182] The SKO virus was mixed 1:1:1 (0.5ml each) and infected into a total of 20,000 fibroblasts in one well of a twelve-well plate, at 37 degrees, 5% CO 2 The conditions were cultured in mES medium, mES supplemented with vitamin C, KSR-BN medium and Basal3.0 medium. On the 10th day after infection, the number of reprogrammed clones was directly counted under a fluorescent microscope, and the ratio of reprogrammed cells was analyzed by flow cytometry. figure 1 a is the number of reprogramming clones induced by every 20,000 Oct4-GFP transgenic embryonic fibroblasts on the 10th day after infection under the culture conditions of mES and mES supplemented with vitamin C, KSR-BN and Basal3.04; The number of repetitions in each experimental group is n=3, and the error bars represent standard deviations. The results showed that on the 10th da...

Embodiment 2

[0186] Embodiment 2: Improvement to Basal3.0

[0187] During the Basal3.0 concentration test, the significant effect of lithium chloride on reprogramming led us to speculate that the inhibition of glycogen synthesis kinase may be the reason for its promotion of reprogramming. Therefore, a series of glycogen synthesis kinase inhibitor substances include CHIR99021, BIO , SB31xxxx were used as candidate substances for the above iPS test experiments (the number of reprogrammed clones and the ratio of reprogrammed cells were used as evaluation criteria for reprogramming efficiency). As a result, the GSK3-β inhibitor CHIR99021 significantly improved the reprogramming efficiency of Basal3.0, so the improved Basal3.0 was added with the glycogen synthesis kinase 3 (GSK3-β) inhibitor CHIR99021, but not limited to adding the chemical composition of CH99021 The defined medium was named iCD1.

[0188] Through the above-mentioned iPS efficiency test experiment, we compared the reprogrammin...

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Abstract

The invention provides a culture system which contains defined components and efficiently acquires induced pluripotent stem cells (iPS cells) from somatic cells. The culture medium additive provided by the invention contains vitamin C, vitamin B12, insulin, glycogen synthase kinase-3 inhibitor, receptor tyrosine kinase and antioxidant. The culture medium additive provided by the invention can also contain a substituted serum cell growth promoter. The invention also provides a complete medium acquired by inducing pluripotent stem cells, which is prepared from one or more of basic culture medium, serum and substituted serum additive and the culture medium additive. The culture system provided by the invention does not have serum, has no animal source pollution, contains defined chemical components, efficiently acquires iPS cells, can maintain cell growth and proliferation in the process of conversion from somatic cells to iPS cells under the condition that feeder cells do not exist, simultaneously accelerates the induction course of the iPS cells obviously, and greatly improves the efficiency of inducing the somatic cells into the iPS cells.

Description

technical field [0001] The invention belongs to the field of cell culture medium, and in particular relates to a culture medium additive which can be used for culturing induced pluripotent stem cells or mammalian cells. Background technique [0002] In 2008, Japanese scientist Yamanaka and his colleagues successfully reprogrammed mouse fibroblasts into embryonic stem cells using four transcription factors (Oct4, Kif4, Sox2, c-Myc), a technique called induced pluripotent stem cells (iPS) technology. Later, fibroblasts from humans and other species such as rats, monkeys, and pigs were also successfully reprogrammed. This technology is considered to be the most important breakthrough in regenerative medicine. Almost all mouse reprogramming processes currently use culture systems that contain serum. The use of the serum culture system has the following disadvantages: 1. The reprogramming process is slow. Serum has been proven to contain some substances that can inhibit reprog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N5/07
CPCC12N2501/33C12N5/0696C12N2506/1307C12N2501/999C12N2500/99C12N2510/00C12N2501/11C12N2501/115C12N2500/38C12N2501/727C12N2501/603C12N2500/90
Inventor 裴端卿陈捷凯刘晶
Owner 杭州健崃生物科技有限公司
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