Novel neural stem cell culture additive, screening method and application of additive and culture medium utilizing additive

A technology for neural stem cells and a screening method, applied in the field of cell culture additives, can solve the problems of undiscovered patent publications, etc., and achieve the effects of promoting neural stem cell proliferation, simple formula and scientific design

Active Publication Date: 2015-06-10
斯坦姆(天津)生物技术研究有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Through the search, no patent publications related to the patent application of the present invention have been found

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  • Novel neural stem cell culture additive, screening method and application of additive and culture medium utilizing additive
  • Novel neural stem cell culture additive, screening method and application of additive and culture medium utilizing additive

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Culture of brain and spinal cord neural stem cells and neural precursor (mother) cells derived from human embryos.

[0060] 1) After strict inspection and screening to exclude common infectious diseases and obtaining the written permission of pregnant women, the embryos between 8 and 10 weeks of gestation were obtained from induced abortion, and the cerebral cortex and spinal cord tissue were dissected under a dissecting microscope.

[0061] 2) The nerve tissue was washed 3 times in DMEM, and digested at 37°C for 45 minutes in a solution of 10 units / ml papain plus 4 mg / ml DNase.

[0062] 3) After adding 4 mg / ml DNase, pipette gently for 5-7 times to disperse the tissue;

[0063] 4) Centrifuge at 1000 rpm for 5 minutes, resuspend in Hanks Balanced Salt Solution (HBSS) without calcium and magnesium, and wash. After repeating three times, the prepared single cell suspension was stained with trypan blue and the cell density was measured with a hemocytometer;

[0064] 5) P...

Embodiment 2

[0067] Neural stem and neural precursor (mother) cells in organ culture (aka tissue culture) adult spinal cord tissue

[0068] 1) After excluding common infectious diseases and obtaining the written permission of the family members of the deceased, the donor spinal cord was dissected, and the spinal cord was cut transversely to a thickness of 2-3 mm, and then the nerve tissue was further cut into 2-3 mm3 tissue blocks under a dissecting microscope.

[0069] 2) After washing with PBS for three times, the complete medium of neural stem cells containing new simplified culture additives was added. Transfer to an uncoated plastic dish at 37 °C, 5% CO 2 After 7 days of culture in a water-jacketed carbon dioxide incubator, it can be digested with papain, dispersed by blowing and blowing to prepare adherent dispersed cell culture. It can also be directly fixed with formalin, and then sectioned for immunohistochemical staining.

Embodiment 3

[0071] Passage dispersed cell culture of mammalian neural stem cells and neural precursor (mother) cells of all embryonic and adult origin.

[0072] 1) After washing and dispersing the cells in calcium-magnesium-free Hanks balanced salt solution (HBSS) for 3 times, digest in 10 units / ml papain plus 4 mg / ml DNase solution at 37°C for 10 minutes;

[0073] 2) After adding 4 mg / ml DNase, pipette gently for 5-7 times to disperse the tissue;

[0074] 3) Centrifuge at 1000 rpm for 5 minutes, resuspend in Hanks Balanced Salt Solution (HBSS) without calcium and magnesium, and wash. After repeating three times, the prepared single cell suspension was stained with trypan blue and the cell density was measured with a hemocytometer;

[0075] 4) Press 5×10 4 The density per square centimeter suspends the cells in the complete medium of neural stem cells containing the new simplified culture supplement, and inoculates them in multi-well culture plates or culture flasks. Cell culture vesse...

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Abstract

The invention provides a novel neural stem cell culture additive. The novel neural stem cell culture additive comprises the following components in final use concentration: 1mg / ml of bovine serum albumin, 50ng / ml of biotin, 1mg / ml of L-carnitine, 0.5mcg / ml of ethanol amine, 7.5mcg / ml of D(+)-galactose, 0.1 micromole of glutathione and 30nmol of sodium selenite. The novel neural stem cell culture additive is based on a scientific design, is simple in formula, low in cost, economic and reasonable, the multiplication of neural stem cells can be promoted, and therefore, the differentiation from the neural stem cells to gliocyte is reduced; the novel neural stem cell culture additive can be applied to aspects of culture of neural stem cell, particularly of the culture of neural stem cells and neural precursor cells of all mammals including human beings and is extremely wide in use range.

Description

technical field [0001] The invention belongs to the field of cell technology, and relates to a cell culture additive, in particular to a novel neural stem cell culture additive, a screening method, an application and a culture medium using the additive. Background technique [0002] Stem cells (stem cells) are primitive undifferentiated cells in the body, while neural stem cells (neural stem cells, NSCs) and neural precursor (mother) cells (neural precursor cells, NPCs, primary differentiated neural stem cells) are stem cell. Because of its promising use in the treatment of neurological diseases and injuries, research and clinical applications have grown by leaps and bounds in recent years. The most important thing for the application of neural stem system is to be able to successfully culture and obtain proliferating neural stem cells in vitro. Because the nerve is the most complex and highly differentiated tissue in the body, the culture of neural stem cells is extremely...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797C12Q1/68C12Q1/06G01N33/569
Inventor 阎军王志梁任峰李遒昶肖敏
Owner 斯坦姆(天津)生物技术研究有限公司
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