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Composition for embryonic stem cell culture and application thereof

A technology of embryonic stem cells and cell culture, applied in embryonic cells, germ cells, animal cells, etc., can solve the problem of not being able to maintain the characteristics of embryonic stem cells for a long time

Active Publication Date: 2015-11-18
LANNUO BIOTECH WUXI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] We added GSK / 3 signaling pathway inhibitor CHIR99021 and MEK inhibitor PD0325901 to the above cell culture medium, and found that although rabbit embryonic stem cells can transiently form stem cell clones in this medium, they cannot maintain embryonic stem cells for a long time characteristics

Method used

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  • Composition for embryonic stem cell culture and application thereof
  • Composition for embryonic stem cell culture and application thereof
  • Composition for embryonic stem cell culture and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Experiment 1: A control experiment of raising rabbit embryos in medium with or without bFGF supplementation.

[0054] The basal culture medium of rabbit embryonic stem cells is KnockOutDMEM (Invitrogen, USA) supplemented with 20% fetal bovine serum, 0.1mM non-essential amino acids, 0.1mM β-mercaptoethanol (Millipore, USA), 4mM glutamine, 50 units / mL penicillin , 50 μg / mL streptomycin.

[0055] Add 10 ng / mL LIF (Millipore, USA) and 10 ng / mL bFGF (Invitrogen, USA) to the basal culture medium, hereinafter referred to as FL culture system.

[0056] Add 10 ng / mL LIF (Millipore, USA), 10 ng / mL bFGF (Invitrogen, USA), 10 μM Y27632, 100 ng / mL Noggin to the basal culture medium, hereinafter referred to as iFLY culture system.

[0057] Only LIF was added to the basal culture medium without bFGF as the control group (no bFGF).

[0058] New Zealand rabbits (purchased from Nanjing Jinling Breeding Rabbit Farm) were treated with superovulation, that is, a total of 2.4 mg of follicl...

Embodiment 2

[0086] Experiment 1': Establishing a rabbit embryonic stem cell line using the iFLY culture system.

[0087] The composition of the iFLY culture system is the same as above. As shown in Figure 2A, after the embryos were cultured in iFLY, stem cell-like growth occurred two days later (D2), and the cells grew more densely after 5 days, and the morphology showed the characteristics of stem cells (D5). After one passage (P1), the induced stem cells produced obvious stem cell morphology ( Figure 2A ). In addition, experiments have shown that both fresh and frozen blastocysts can be cultured in the iFLY culture system to obtain rabbit embryonic stem cells (10.2% vs. 16.9%), as shown in Table 5. After the frozen embryos at the 1-2 cell stage were thawed and then cultured into blastocysts, the efficiency of rabbit embryonic stem cell induction dropped to 1.5%, as shown in Table 5.

[0088] Table 5 Effects of fresh embryos and frozen embryos on the acquisition of rabbit embryonic st...

Embodiment 3

[0107] Experiment 1'': rabbit embryonic stem cell line #7-1, M5 blastocyst injection experiment.

[0108] Rabbit embryonic stem cell line #7-1 (iFLY system) and stem cell line M5 (FL system) were used for blastocyst injection to generate chimeric animals. Blastocysts of wild-type New Zealand rabbits were used as recipient embryos for cell injection, #7-1 and M5 cells were infected with GFP DNA cells using the green fluorescent (GFP) Fugene conversion system (Roche, USA), so that the stem cell genome carried the GFP gene, and GFP protein is expressed in cells; under a fluorescent microscope, it shows green fluorescence. Cells from a successfully infected GFP cell line (#7-1, M5) were microinjected into wild-type blastocysts, and approximately 5-10 stem cells were injected into each blastocyst. About 10-20 injected blastocysts were transplanted into estrus-synchronized female rabbits, and the ears of newborn rabbits were sampled for PCR detection of GFPDNA. At the same time, e...

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Abstract

The invention provides a composition for embryonic stem cell culture. The composition can guarantee that embryonic stem cells are in an undifferentiated state for a long term and have the ability of infinite proliferation. The composition comprises leukaemia inhibitory factors (LIF), basic fibroblast growth factor (bFGF), Rho kinase inhibitor Y27632 and bone morphogenetic protein inhibitor Noggin. The invention further provides cell culture liquid containing the composition and application of the composition.

Description

technical field [0001] The invention relates to the field of cytology, in particular to a composition for culturing embryonic stem cells and its application. Background technique [0002] The successful isolation and culture of human embryonic stem cells is an important progress in today's life sciences. It is isolated from the inner cell mass of early human preimplantation embryos and can maintain undifferentiated and continuously proliferate under appropriate conditions in vitro. kind of stem cells. Theoretically, it has the potential to differentiate into all cell tissues of the body, and is called "seed cell". People can use different culture conditions to induce differentiation of embryonic stem cells to produce a large number of different types of cells as cell donors for cell transplantation, tissue replacement and even organ cloning, providing unlimited cell sources for the future treatment of many intractable diseases in humans. Therefore, it has broad prospects i...

Claims

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Application Information

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IPC IPC(8): C12N5/0735
Inventor 杜福良徐捷陈建宏薛非杨澜安礼友
Owner LANNUO BIOTECH WUXI
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