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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 life span of MEF, and inability to pass on for a long time, so as to maintain self-renewal and rapid value-added, which is important Application value, economic value, and effect of reduction in cultivation cost

Active Publication Date: 2009-09-16
BEIJING WEITONGDA BIOTECH
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  • Abstract
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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] Embodiment 1, using the human umbilical vein endothelial cell line ECV-304 as trophoblast cells to culture mouse embryonic stem cells

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

[0034] 1. ECV-304 cell culture

[0035] Main experimental materials:

[0036] Cells: 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) in high-sugar DMEM (product of Hyclone Company, article number: SH30243.01), so that the mass percentage of calf serum is 10%, so that 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. Recovery of ECV-304 cells:

[0040] (1) Cell recovery: Take out the frozen cells from the frozen liquid nitrogen tank, and quickly place them in a water bath at 37°C-42°C to melt the cells ...

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 Step 1 of Example 1.

[0078] Cells are human bladder cancer epithelial cells T24 (ATCC company of the United States, ATCC no.HTB-4 TM );

[0079] The cell culture medium is T24 cell culture medium: add fetal calf serum and thioglycerol (MTG) in high-sugar DMEM (product of Hyclone Company, article number: SH30243.01), so that the mass percentage of fetal calf 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 were human bladder cancer epithelial cells T24 and the cell culture medium was T24 cell culture medium, other experi...

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. Stemness molecules (marker molecules) of mouse embryonic stem cells: transcription factors Oct3 / 4, Nanog, Sox2.

[0086] 2. RT-PCR reaction primers: reverse transcription synthesis of cDNA using the 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'CCCGTGGTTACCTTCTTCC3', Sox2P2: 5'TTTCTCCAGTTCGCAGTCC3'; internal reference gene β-actin P1: 5'GCTGTCCCTGTATGCCTCT3', β-actin P 5'TTGATGTCACGCACGATTT3'. Wherein, OctP1 and OctP2 are primers for amplifying the transcription factor Oct3 / 4, NanogP1 and NanogP2 ...

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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 cultivating mouse embryonic stem cells and a special culture medium thereof. Background technique [0002] Embryonic stem cells (ES cells), also known as ES cells, are totipotent cells that exist in early embryos before implantation. Totipotency is the ability to differentiate into tissue cells that develop into the three germ layers. Embryonic stem cells can be cultured and passaged in vitro, maintain an undifferentiated diploid state and their totipotency, and have the ability to form chimeric animals. Mouse embryonic stem cells were first isolated and cultured from early mouse embryos by Evans and Kaufman (1981) and Martin (1981) respectively, and cell lines were established. Because of its totipotency, it is widely used in the research of embryo development and embryo engineering. For example, adding different differentiation factors to the culture medium can induce its differentiation and development into c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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