Preparation method of clinical-grade neural stem cell

A technology of neural stem cells and cytokines, applied in nervous system cells, biochemical equipment and methods, animal cells, etc., can solve the problems of inability to quickly obtain neural stem cells with high proliferation ability, low purity of neural stem cells, and imperfect culture system

Active Publication Date: 2018-05-25
JILIN TUO HUA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purity of neural stem cells obtained by ordinary extraction methods is low, and the culture system is not perfect enough to quickly obtain neural stem cells with high proliferation ability and high activity, and it is difficult to meet the requirements of clinical reinfusion

Method used

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  • Preparation method of clinical-grade neural stem cell
  • Preparation method of clinical-grade neural stem cell
  • Preparation method of clinical-grade neural stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Isolation of a single neural stem cell

[0054] The hippocampal tissue of the subject was obtained in the hospital, and the informed consent of the donor’s family members was signed, and various virus tests were negative; the hippocampal and hippocampal dentate gyrus tissues were filled with 5ml of digestive enzymes (accutase and papain in a mass ratio of 1 : 1) in a 15ml centrifuge tube, shake gently, 37°C, 15min;

[0055] After the digestion is complete, add 5ml of basal medium containing antibiotics (Table 1), mix well, and gently pipette;

[0056] BD 45μm membrane filter neural stem cells, 500g, centrifuged for 5min;

[0057] Resuspend the neural stem cell pellet in basal medium containing double antibodies (Table 1), and centrifuge at 500g for 5min;

[0058] Resuspend the neural stem cell pellet in 10ml of complete medium, mix well, and count.

[0059] Table 1. Culture medium composition

[0060]

Embodiment 2

[0061] Example 2: Inoculation and cultivation of neural stem cells

[0062] The concentration of the neural stem cells obtained in Example 1 was adjusted to 5*10 5 / ml;

[0063] Inoculate into ordinary T75 culture flasks (Table 2), 25 to 30ml / T75, place at 37°C, 5% CO 2 Cultivated in a constant temperature incubator;

[0064] After 12 to 24 hours, collect the neural stem cell suspension, centrifuge at 400g for 5min, and remove the supernatant;

[0065] Complete medium (Table 2) was resuspended, mixed, gently pipetted, and counted;

[0066] According to the cell viability and quantity, adjust the concentration of neural stem cells to 1.5*10 5 / ml, inoculated into low adsorption T75 culture flasks (Table 2), 25 to 30ml / T75, placed at 37°C, 5% CO 2 Cultivated in a constant temperature incubator;

[0067] Change the medium in half every four days according to the growth status (the diameter of more than 80% of the neurospheres is not less than 200 μm);

[0068] Digestion (a...

Embodiment 3

[0071] Example 3: Passage of nerve cells

[0072] Subculture when the size of the cell sphere is about 200 μm to 450 μm, and collect the cell sphere culture medium;

[0073] Centrifuge at 200g for 5min, remove the supernatant, and keep the pellet;

[0074] Add 2ml of mixed digestion solution, gently blow off the neural stem cell pellet, and shake in a water bath at 37°C for 5 minutes;

[0075] Gently pipette the neural stem cell suspension, and observe the state of the cell sphere under a microscope. If it is not completely digested, continue to shake it in a water bath at 37°C for 5 minutes;

[0076] Add 10ml of basal medium (Table 3), gently pipette the neural stem cell suspension, and filter the neural stem cells with a BD 45 μm filter membrane;

[0077] Centrifuge at 500g for 5min, resuspend with complete medium (same as above), and count;

[0078] Adjust neural stem cells to 1.5*10 5 / ml, inoculated into low-adsorption T75 culture flasks (Table 3), 25 to 30ml / T75, pla...

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Abstract

The disclosure provides a preparation method of a clinical-grade neural stem cell. The preparation method comprises the following steps of a), providing a single neural stem cell; b), inoculating theneural stem cell into a conventional culture flask, and subsequently replacing with a low-adsorption culture flask; c), adding a tumor necrosis factor-alpha, albumin, B27 and an animal-free protogonocyte factor into a culture medium to stimulate the multiplication of the neutral stem cell; d), carrying out half-amount medium change according to a growth status; e), carrying out digestion and passage according to the growth status; f), harvesting to obtain a high-purity, high-activity and further high-differentiation-potential clinical-grade neural stem cell. The method provided by the disclosure is simple to operate; the obtained cell is high in purity and further high in multiplication capacity, and the neutral stem cell prepared through the method provided by the disclosure meets a clinical-grade requirement.

Description

technical field [0001] The present disclosure relates to the field of biotechnology, in particular to an efficient method for preparing clinical-grade neural stem cells. Background technique [0002] Neural stem cells originate from embryonic stem cells and adult stem cells, and exist in the telencephalon, cerebellum, hippocampus, striatum, cerebral cortex, ventricle / subventricular zone, ependyma / subventricular zone, spinal cord and adult brain of the embryonic brain subventricular zone, striatum, hippocampus dentate gyrus, spinal cord, etc. [0003] Neural stem cells have the ability of self-renewal and multi-lineage differentiation potential to neurons, astrocytes and oligodendrocytes throughout life. Its discovery and research is one of the most important advances in the field of neurobiology. [0004] In addition to the two basic characteristics of self-renewal and multi-lineage differentiation potential, neural stem cells also have transdifferentiation (plasticity), u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797
CPCC12N5/0623C12N2501/105C12N2501/11C12N2501/13C12N2501/115C12N2501/998C12N2501/25
Inventor 李超姜丽君毕薇薇
Owner JILIN TUO HUA BIOTECH
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