Neural stem cells medium and method for performing human neural stem cells in-vitro long-term culture and amplification by using neural stem cells medium

A neural stem cell and culture medium technology, applied in the field of neural stem cell culture, can solve the problems of mixed sources of neural stem cells, difficulty in obtaining neural stem cells, lack of embryonic or fetal sources, etc.

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

AI Technical Summary

Problems solved by technology

[0003] The number of neural stem cells cultured by the conventional method of digesting and passing neural stem cells with trypsin is small, and it is difficult to obtain a large number of neural stem cells for cli

Method used

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  • Neural stem cells medium and method for performing human neural stem cells in-vitro long-term culture and amplification by using neural stem cells medium
  • Neural stem cells medium and method for performing human neural stem cells in-vitro long-term culture and amplification by using neural stem cells medium
  • Neural stem cells medium and method for performing human neural stem cells in-vitro long-term culture and amplification by using neural stem cells medium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 The culture expansion of neural stem cells

[0055] (1) Neural stem cell culture: Take the prepared fetal brain-derived hippocampal neural stem cells, press 1-2x10 6 / ml cells were grown in cell culture flasks. Neural stem cell culture medium was used for culture and expansion, and the cell bottle was placed in 5% CO 2 , 37 ℃ temperature incubator for cultivation. According to the condition of cell proliferation, half of the culture medium is generally changed every 3-4 days. The components of the neural stem cell culture medium are:

[0056]

[0057] The preparation method of this neural stem cell culture medium is: take 12gDMEM / F12 culture medium and mix with 1000mL sterile deionized water, add 4-hydroxyethylpiperazine ethanesulfonic acid and stir evenly to make basic culture medium, make the pH value of the solution be controlled at 7.0~7.1; then add the remaining components and mix well.

[0058] (2) The passage expansion method is as follows: t...

Embodiment 2

[0059] Example 2 Nestin Identification of Neural Stem Cells

[0060] Neural stem cell-specific antigen nestin (nestin) is a specific antigen commonly used to dispose of neural stem cells and other stem cells. Immunofluorescence staining with nestin (nestin) antibody was used to preliminarily identify neural stem cells.

[0061] Neural stem cell spheres cultured continuously for 10 months were directly planted on poly-lysine-coated glass coverslips at a cell density of 5×10 4 5-6 days later, immunofluorescent staining was performed with nestin antibody.

[0062] ① Use a pipette to take 3-5 neural stem cell sphere suspensions that have been cultured for 10 months and drop them on the poly-lysine-coated slides, and place them in a 37°C incubator for 24 hours to make the cells adhere to the slides.

[0063] ② Fix with 4% paraformaldehyde + 0.3% glutaraldehyde for 15 minutes.

[0064] ③ After washing with DPBS buffer for 3 times, block with DPBS containing 5% normal goat serum (c...

Embodiment 3

[0068] Example 3 Differentiation and Identification of Neural Stem Cells

[0069] Neural stem cells can differentiate into neurons, astrocytes and oligodendrocytes. Neurons contain β-TublinIII antigen, and the cells are fluorescently labeled with β-TublinIII antibody to detect whether they are neurons. Astrocytes contain GFAP antigen protein, and the cells are fluorescently labeled with GFAP antibody to detect whether they are astrocytes. The oligodendrocytes contain (Gal-C) antigen protein, and the cells are fluorescently labeled with Gal-C antibody to detect whether they are oligodendrocytes.

[0070] experimental method

[0071] (1) The neural stem cell spheres cultured for 10 months were directly pipetted or digested with 0.125% trypsin and 0.5mMEDTA·4Na solution to make a single cell suspension.

[0072] (2) Cells in 5×10 4 Seed on polylysine (100mg / ml)-coated glass coverslips per slice density, add 2% fetal bovine serum (FBS) to DMEM / F12 medium, B27 additive (1:50 di...

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Abstract

The invention relates to a neural stem cells medium and a method for performing human neural stem cells in-vitro long-term culture and amplification by using the neural stem cells medium. The neural stem cells medium comprises the following ingredients by weight proportion: 100-1000 micrograms of heparin sodium, 10-100 micrograms of vitamin E, 5-50 milligrams of insulin human recombinant, 0.5-5 milligrams of putrescine, 2-10 micrograms of sodium selenite, 2-10 milligrams of human transferrin, 2-10 micrograms of progestin, 300 milligrams of L-glutamine, 5.9 grams of 2-[4-(2-Hydroxyethyl)-1-piperazine]ethanesulfonic acid, 10-100 micrograms of recombinant human epidermal growth factors, 10-100 micrograms of recombinant human basic fibroblast growth factors, 20-200 milligrams of vitamin C glucoside and 40,000-400,000 IU (international unit) of gentamicin. By the neural stem cells medium, the technical problems that human neural stem cells are easy to differentiate when cultured in vitro and long-term culture and amplification are difficult to implement are solved.

Description

technical field [0001] The invention relates to a method for culturing neural stem cells, in particular to a culture medium for neural stem cells and a long-term culture and expansion method for human neural stem cells in vitro. Background technique [0002] In 1992, Reynolds and Weiss discovered that mammalian forebrain has neural stem cells, which can be cultured and expanded in vitro. Neural stem cells have the following characteristics: 1 self-renewal ability; 2 differentiation potential: they have multi-directional differentiation potential and can differentiate into three types of cells including neurons, astrocytes and oligodendrocytes. Neural stem cells have direct or indirect therapeutic effects on nervous system damage and neurodegenerative diseases, so mass culture and expansion of human neural stem cells has great application value. [0003] The conventional method of cultured neural stem cells by trypsinization is small, and it is difficult to obtain a large nu...

Claims

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Application Information

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IPC IPC(8): C12N5/0797
Inventor 陈世明章瑾宣丹英王卓然
Owner ZHEJIANG ORIGIN BIOTECH
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