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Method for preparing and separating definitive endoderm

A technology for definitive endoderm and embryonic stem cells, which is applied in the field of preparation and isolation of definitive endoderm cells, and can solve problems such as the inability to better distinguish VE and DE

Active Publication Date: 2010-03-31
引加苏州生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the development of mammals, VE comes from primitive endoderm (Primitive endoderm, PE) and does not participate in organ development, but VE and DE co-express many of the same genes, and VE is easily induced, and the previous induction methods are not good. Differentiate between VE and DE, often resulting in a mixed cell population

Method used

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  • Method for preparing and separating definitive endoderm
  • Method for preparing and separating definitive endoderm
  • Method for preparing and separating definitive endoderm

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Experimental program
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preparation example Construction

[0078] The present invention also includes the cell group containing definitive endoderm cells obtained by the aforementioned preparation method. In the cell population, the number of definitive endoderm cells accounts for more than 60% of the total number of cells, preferably more than 70% of the total number of cells; more preferably more than 80% of the total number of cells.

[0079] Using FACS and surface marker technology, it is possible to enrich definitive endoderm cells into Cxcr4+ / Cd140α- cell population or Cxcr4+ / EpCam+ cell population; use RT-PCR and other gene expression analysis techniques to prove the purity of the enriched endoderm cells Higher, less contamination from other germ layer cells.

[0080] Therefore, the present invention also includes cell populations with higher content of definitive endoderm cells separated by sorting techniques. Preferably, the cell population is a cell population positive for Cxcr4 molecules on the cell surface and positive fo...

Embodiment 1

[0106] Embodiment 1. Preparation of culture medium

[0107] 1. Preparation of serum-free medium

[0108] Prepare the mixture of IMDM and F12 according to the dosage shown in Table 1:

[0109] Table 1

[0110] IMDM

75% (volume ratio)

[0111] (available from Invitrogen; Cat# 12440-053, Gibco)

F12 mixture

(available from Invitrogen; Cat#: 11765-054, Gibco)

25% (volume ratio)

[0112] In the previously prepared mixture, add the ingredients of the formula in Table 2:

[0113] Table 2

[0114] N2

(available from Invitrogen, Cat: 17502-048)

0.5% (volume ratio)

B27 (Vitamin A deficiency)

(available from Invitrogen, Cat: 12587-010)

1% (volume ratio)

BSA

0.5mg / ml

penicillin or streptomycin (Gibco)

1% (volume ratio)

Vitamin C (Vc)

0.5mM

3-Mercaptothioglycerol (MTG)

4.5×10 -4 M

[0115] 2. Low serum culture medium

[0116] In GME...

Embodiment 2

[0127] Example 2. Inducing mouse embryonic stem cells to differentiate into definitive endoderm cells and screening using Cxcr4+ / c-Kit+ markers

[0128] The method for inducing mouse embryonic stem cells to differentiate into definitive endoderm cells (DE) using the medium prepared above comprises:

[0129] 1) Mouse ES cells (purchased from ATCC, CRL-1821) were cultured in low serum to maintain an undifferentiated state;

[0130] 2) The mouse ES cells were digested with 0.25% Trypin-EDTA (Gibco), counted, according to 2×10 4 Individuals / ml with a total volume of 15ml were planted in a 10cm low-adherence bacterial culture dish, and the serum-free culture prepared above was used to culture under normal conditions for 48 hours to form embryoid bodies;

[0131] 3) Collect the embryoid bodies and continue to induce them with differentiation medium 6 for 48 hours.

[0132] 4) The induced embryoid bodies were digested into single cells, labeled with specific antibodies, and used at...

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Abstract

The invention discloses a method for preparing and separating definitive endoderm (DE); the invention also discloses a differential medium used for promoting cytotropism differentiation of DE; the differential medium contains activin A and Wnt signal channel agonist; and the invention also discloses a surface marker used for sorting endoderm.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a method for preparing and isolating definitive endoderm cells (Definitive Endoderm, DE). Background technique [0002] The embryonic stem cells (Embryonic Stem Cells, ES) derived from the cell mass in the blastocyst stage are pluripotent, and under appropriate culture conditions, they can maintain a nearly uniform and theoretically unlimited self-renewal ability; while under specific induction Under certain conditions, they can differentiate into almost all somatic cells. Embryonic stem cells provide good conditions for modern life science and pharmaceutical science research. They are an ideal model for studying the early embryonic development of mammals. They also play a role in cell-based biological therapy (Cell-Based Therapy), drug screening, and toxicological testing. to an important role. [0003] In recent years, the further understanding of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C07K16/28G01N33/53
Inventor 王欣丁小燕李福明
Owner 引加苏州生物医药科技有限公司
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