Human spinal cord neural stem cell induced differentiation culture system and method

A technology for spinal nerves and induction of differentiation, applied in the field of biomedicine, can solve the problems of lack of similar research technologies, no patented technology release, etc., and achieve the effect of high differentiation purity

Pending Publication Date: 2022-03-22
ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is even a lack of similar research technologies in Chi

Method used

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  • Human spinal cord neural stem cell induced differentiation culture system and method
  • Human spinal cord neural stem cell induced differentiation culture system and method
  • Human spinal cord neural stem cell induced differentiation culture system and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The preparation of embodiment 1 culture medium

[0046] 1. N2B27 medium (take the configuration of 500mL N2B27 medium as an example):

[0047]

[0048]

[0049] In this medium, penicillin-streptomycin double-antibody and GlutaMAX supplement can be selectively added according to needs, so as to prevent the culture medium from being contaminated by bacteria, and the culture with lower ammonia content can show higher cell viability by adding a small amount of GlutaMAX supplement.

[0050] 2. Spinal cord neural stem cell induction medium (take the preparation of 10mL spinal cord neural stem cell induction medium as an example)

[0051]

[0052] Wherein, in the spinal cord neural stem cell induction medium, specific inhibitors and growth factor signaling proteins are added to achieve the purpose of regulating the proliferation and differentiation of specific types of cells.

[0053] 3. Spinal cord neural stem cell maintenance medium (take 50ml spinal cord neural st...

Embodiment 2

[0058] Example 2 Subculture of human embryonic stem cells (taking H9 cells as an example, where H9 cells were purchased from Guangzhou Guiyi Biotechnology Co., Ltd.)

[0059] Recovery of H9 cells

[0060] One hour before cell recovery, Matrigel was used to coat the culture dish to improve cell attachment efficiency. Specifically, 1ml of Matrigel solution was used for each well of a 6-well plate (160μL of Matrigel was dissolved in 12mL of DMEM pre-cooled on ice. / F12 medium) plated, placed at room temperature for more than 1h, ultra-clean bench UV disinfection; at the same time, DMEM / F12 medium, mTeSR TM 1 Take the culture medium out of the refrigerator and preheat it in a 37°C water bath;

[0061] Take out the H9 cells frozen in liquid nitrogen, and immediately put them in a 37°C water bath; after they are completely thawed, mix the H9 cells into 5 mL of warm DMEM / F12 medium, centrifuge at 800r for 5 minutes, and remove the supernatant solution; with 2mL mTesR TM 1 Gently...

Embodiment 3

[0066] Example 3 Induction of differentiation of H9 cells

[0067] The H9 cells subcultured in Example 2 were replaced with the mTeSR1 medium the next day, and replaced with the spinal cord neural stem cell induction medium in Example 1 when the H9 cells grew to 50% confluence. During the culture period, depending on the cell growth density, passage 1-2 times, and use Y27632 to increase the survival rate of single cells. The specific passage method is the same as that in Example 2.

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Abstract

The invention discloses a human spinal cord neural stem cell induced differentiation culture system and method.The human spinal cord neural stem cell induced differentiation culture system mainly comprises a spinal cord neural stem cell induction culture medium and a spinal cord neural stem cell maintenance culture medium, and the spinal cord neural stem cell induction culture medium takes an N2B27 culture medium as a basic culture medium; every 10 ml of the N2B27 culture medium comprises 10 to 12 [mu] l of a BMP inhibitor, 10 to 12 [mu] l of an ALK5 inhibitor, 10 to 12 [mu] l of a GSK-3 inhibitor, 10 to 12 [mu] l of FGF2 and 10 to 12 [mu] l of FGF8. The spinal cord neural stem cell maintenance culture medium takes an N2B27 culture medium as a basic culture medium, and every 50 ml of the N2B27 culture medium comprises 10-12 [mu] l of an ALK5 inhibitor, 15-18 [mu] l of a GSK-3 inhibitor and 1-1.2 [mu] l of a Hedgehog agonist. The induced differentiation culture system is used for carrying out induced differentiation culture on the human embryonic stem cells, the human spinal cord NSC cells can be directionally differentiated and formed, and the human spinal cord neural stem cells have multidirectional differentiation potential, so that the human spinal cord development/regeneration related cell process can be better understood; the method is of great significance to follow-up treatment strategies for diseases such as human spinal cord injury and degeneration.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, specifically relates to a human spinal cord neural stem cell induced differentiation culture system, and also relates to a human spinal cord neural stem cell induced differentiation method based on the human spinal cord neural stem cell induced differentiation culture system. Background technique [0002] Neural stem cells (Neural Stem Cells, NSCs) are a cell population that exists in the nervous system and has the potential to differentiate into neurons, astrocytes and oligodendrocytes, and is capable of self-renewal. Human embryonic stem cells (Humanembryonic stem cells, HESCs) can be induced to differentiate in vitro to form NSCs, so they have become an important platform for research on stem cell therapy. HESC has the characteristics of self-renewal (Self-renewal) and pluripotency (Pluripotent), and can differentiate into three germ layers (endoderm, mesoderm and ectoderm) in vitro and th...

Claims

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Application Information

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IPC IPC(8): C12N5/0797C12N5/0735
CPCC12N5/0623C12N2501/155C12N2501/15C12N2501/727C12N2501/115C12N2501/119C12N2501/40C12N2501/91C12N2501/11C12N2501/999C12N2506/02
Inventor 刘超陈雪莹刘丽华
Owner ANHUI MEDICAL UNIV
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