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Method for culturing mouse embryo stem cell and its dedicated culture medium

A technology of embryonic stem cells and mouse embryos, applied in embryonic cells, germ cells, tissue culture, etc., can solve the problems of lack of scientific research funds, limited lifespan of MEF, complicated operation, etc., to maintain self-renewal and rapid value-added, important applications Value and economic value, simple effect of cultivation ingredients

Active Publication Date: 2007-07-11
BEIJING WEITONGDA BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the past 20 years since the establishment of the mES line, although the in vitro culture technology of mouse embryonic stem cells is the most mature, the above-mentioned culture system still has the following disadvantages: (1) The operation is complicated: the mES in vitro culture process includes the preparation of 12.5-14.5-day pregnant mice , MEF isolation and culture, MEF trophoblast cell preparation, conditioned medium preparation, mES passage, etc.
(2) MEFs have a limited lifespan and cannot be passaged in vitro for a long time
(4) During the culture of embryonic stem cells, the chromosome release of dead MEF cells may cause mutations in embryonic stem cells and affect the maintenance of normal karyotype
(5) The composition of the culture medium is complex: In the mES culture system, in addition to the basic medium DMEM and fetal bovine serum, β-mercaptoethanol (β-ME) or thioglycerol, glutamine, non-essential amino acids, recombinant the LIF
At the same time, long-term storage of amino acid components and recombinant LIF will easily lead to loss of biological activity
(6) In the traditional culture method, pregnant mice, various amino acids, artificial recombinant LIF and other biological materials and reagents with very high cost are used, which greatly restricts the research work related to mES due to the relative lack of scientific research funds in my country.

Method used

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  • Method for culturing mouse embryo stem cell and its dedicated culture medium
  • Method for culturing mouse embryo stem cell and its dedicated culture medium
  • Method for culturing mouse embryo stem cell and its dedicated culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Culturing mouse embryonic stem cells with human umbilical vein endothelial cell line ECV-304 as trophoblast cells

[0033] 1. Preparation of ECV-304 trophoblast cells

[0034] 1. ECV-304 cell culture

[0035] Main experimental materials:

[0036] Cell: human umbilical vein endothelial cell line ECV-304 (Japan DSMZ Company, DSMZ no. ACC310), hereinafter referred to as ECV-304.

[0037] Cell culture medium: add calf serum and thioglycerol (MTG) to high-glucose DMEM (product of Hyclone company, product number: SH30243.01), so that the mass percentage of calf serum is 10%, and the thioglycerol The content of glycerin is 1.5×10 -4 mol / L.

[0038] Digestion solution: a solution obtained by adding 0.25% trypsin to 0.02% EDTA at a volume ratio of 1:1.

[0039] 2. ECV-304 cell recovery:

[0040] (1) Cell recovery: Take out the cryopreserved cells from the cryopreservation liquid nitrogen tank, quickly place them in a 37°C-42°C water bath to rapidly thaw the cell...

Embodiment 2

[0075] Example 2. Using human bladder cancer epithelial cells T24 as trophoblast cells to culture mouse embryonic stem cells

[0076] 1. Preparation of T24 trophoblast cells

[0077] Except for the cells and cell culture medium in the experimental materials, other experimental materials and experimental methods are the same as in step 1 of Example 1.

[0078] The cells are human bladder cancer epithelial cells T24 (American ATCC company, ATCC no.HTB-4 TM );

[0079] The cell culture medium is T24 cell culture medium: add fetal bovine serum and thioglycerol (MTG) to high-glucose DMEM (product of Hyclone company, product number: SH30243.01), so that the mass percentage of fetal bovine serum is 20%. %, so that the content of thioglycerol is 3.0×10 -4 mol / L.

[0080] 2. In vitro culture of established embryonic stem cells

[0081] Except that the trophoblast cells are human bladder cancer epithelial cells T24, and the cell culture medium is T24 cell culture medium, other expe...

Embodiment 3

[0083] Example 3. Detection of mES stemness marker molecules cultured with ECV-304 or human bladder cancer epithelial cells T24 as trophoblast cells.

[0084] 1. Reverse transcription polymerase chain reaction (RT-PCR) to detect the expression of mES stemness molecular markers at the mRNA level:

[0085] 1. Mouse embryonic stem cell stemness molecules (marker molecules): transcription factors Oct3 / 4, Nanog, Sox2.

[0086] 2. RT-PCR reaction primers: reverse transcription synthesis of cDNA using universal primer OligodT, PCR amplification of the above gene primer sequences are: OctP1: 5'AATGCCGTGAAGTTGGAG3', OctP2: 5'GAAGCGACAGATGGTGGT3'; NanogP1: 5'GCCCTGATTCTTCTACCA3', NanogP2: 5'AGATGCGTTCACCAGATAG3'; Sox2P1: 5'CCCGTGGTTACCTCTTCC3', Sox2P2: 5'TTCTCCAGTTCGCAGTCC3'; internal reference gene β-actin P1: 5'GCTGTCCCTGTATGCCTCT3', β-actin P2: 5'TTGATGTCACGCACGATTT3'. Among them, OctP1 and OctP2 are primers for amplifying transcription factor Oct3 / 4, NanogP1 and NanogP2 are primers...

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Abstract

The invention discloses a mouse embryo stem cell culturing method and specific cultivating base, which cultures embryo stem cell on the trophoblast cell, wherein the trophoblast cell is epithelial cell or endothelium cell; the cultivating base consists of epithelial cell or endothelium cell and culturing liquid of embryo stem cell, which is high-sugar DMEM with 10-20% embryo cow serum or calf serum with 1. 5-3. 0*10-4mol / L Monothioglycerol, MTG.

Description

technical field [0001] The invention relates to a method for culturing mouse embryonic stem cells and a special medium thereof. Background technique [0002] Embryonic stem cells (ES cells), also known as ES cells, are totipotent cells that exist in early preimplantation embryos. Totipotency is the ability to differentiate into tissue cells of the three germ layers. Embryonic stem cells can be cultured and passaged in vitro, maintain undifferentiated diploid state and totipotency, and have the ability to form chimeric animals. Mouse embryonic stem cells were firstly isolated and cultured from early mouse embryos by Evans and Kaufman (1981) and Martin (1981), respectively, and established cell lines. Because of its totipotency, it is widely used in embryonic development and embryo engineering research. For example, by adding different differentiation factors to the culture medium, it can be induced to differentiate and develop into cardiomyocytes, lymphocytes and nerve cel...

Claims

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Application Information

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IPC IPC(8): C12N5/06C12N5/0735
Inventor 周海胜孙晓萌邓宏魁丁明孝
Owner BEIJING WEITONGDA BIOTECH
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