Application of inorganic nano-material layered double hydroxides (LDHs) in mouse embryonic stem cell culture

An inorganic nanomaterial and stem cell culture technology, applied in embryonic cells, animal cells, vertebrate cells, etc., can solve the problems of cumbersome procedures in the embryonic stem cell culture process, the cost of animal components, and the distance between clinical applications, etc. Effect of contact with cell surface, low cytotoxicity, good biocompatibility

Active Publication Date: 2014-08-06
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the relevant research results of ESC cells have shown broad application prospects in the field of clinical medicine, the basic research of both embryonic stem cells and adult stem cells is far from clinical application, and there are still many difficult problems to solve, such as: The stem cell culture process is cumbersome, with animal components and high costs, which are major problems to be solved at present
[0003] The combination of nanotechnology and stem cells is likely to bring about major breakthroughs

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Preparation of nano-layered double hydroxide (SLW-1)

[0019] Prepare Mg(NO 3 ) 2 ·6H 2 O (1.536 g, 0.006mol) and Al(NO 3 ) 3 9H 2 O ( 0.75 g, 0.002 mol) metal mixed salt solution was 40ml in total, water was used as solvent, and the molar ratio of Mg / Al was 1:1, and 0.016mol NaOH solution was prepared. Under N2 atmosphere, add the metal salt mixed solution into the vigorously stirred NaOH solution, transfer the resulting suspension to a hydrothermal synthesis kettle, and take it out after 18 hours at 100°C to obtain a SLW-1 suspension with a particle size of 100-120nm, vacuum Weigh after drying in the oven. The synthesized SLW-1 was observed by transmission electron microscope (as figure 1 ), with a good crystal structure, all in hexagonal shape.

Embodiment 2

[0020] Example 2: Culture and purification of mouse embryonic stem cells mESC.

[0021] Mouse embryonic fibroblasts (MEF) were obtained from primary generation, cultured to passage 3-5, treated with 10 μg / ml mitomycin C for 3 hours, and used as feeder layer cells within 1 week. Complete medium for mESC: high-glucose DMEM, 15% fetal bovine serum, 0.1 mmol / L non-essential amino acids, 2 mmol / L glutamine, 0.1 mmol / L β-mercaptoethanol, 1000U / ml 0.1 leukemia inhibitory factor, normal After culturing for 2-3 days, add trypsin (0.25%)-EDTA (0.02%) to digest the cells, neutralize the trypsin in the culture medium, place the cells on a 0.1%-coated petri dish, and culture for 30 minutes. After counting the unattached mESCs, they were inoculated at 2×104 per well on a six-well plate coated with 1% gelatin. After 24 hours of culture, they were replaced with culture medium without LIF factor, and different doses of The SLW-1 nanoparticles obtained in Example 1 were co-cultured for 5 days,...

Embodiment 3

[0022] Example 3: Effect of SLW-1 on mESC self-renewal

[0023] (1) Alkaline phosphatase (ALP) activity detection

[0024] An important biological characteristic of undifferentiated embryonic stem cells is the ability to synthesize high concentrations of alkaline phosphatase (AP), which can reflect the differentiation state of embryonic stem cells. Alkaline phosphatase staining was performed on the fixed mESCs obtained in Example 2. Such as figure 2 As shown, morphologically, mESCs depleted of LIF factor have been completely differentiated, while mESCs added with SLW-1 can inhibit differentiation; mESCs treated with final concentration of SLW-1 at 37.5 μg / ml and 75 μg / ml have strong alkaline phosphatase staining Positive, while mESC depleted of LIF factor was weakly positive. It shows that SLW-1 can promote the self-renewal ability of mESC.

[0025] (2) Realtime PCR was used to detect the expression of pluripotency and self-renewal transcription factors in mouse embryonic...

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Abstract

The invention relates to application of inorganic nano-material layered double hydroxides (LDHs) in mouse embryonic stem cell culture. Under the condition that LIFs are not added, the inorganic nano-material LDHs can promote expression of pluripotency genes and restrains cell differentiation, and in addition, the treated cells still have the potency of differentiating to three germ layers. The inorganic nano-material LDHs have excellent biocompatibility, low cytotoxicity, easiness in contact with cell surfaces, low cost, convenience and rapidness in material synthesis and excellent effect, and has extensive application prospect in research of embryonic stem cells.

Description

technical field [0001] The invention relates to the application of a kind of inorganic nano material layered double hydroxide in the culture of mouse embryonic stem cells. Background technique [0002] Embryonic stem cells (ESCs) are derived from the inner cell mass of mammalian blastocysts and have unlimited self-renewal capabilities. These cells are pluripotent stem cells that can differentiate into any cell type in the three germ layers. Because of these characteristics of ESC, ESC has become a hot spot in life science research today. At present, ESC has made breakthroughs in the treatment of diabetes, cardiomyopathy, neurodegenerative diseases, and bone and joint diseases. Although the relevant research results of ESC cells have shown broad application prospects in the field of clinical medicine, the basic research of both embryonic stem cells and adult stem cells is far from clinical application, and there are still many difficult problems to solve, such as: The stem ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735
Inventor 汪世龙孙晓宇朱融融吴又君
Owner TONGJI UNIV
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