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Vitrification cryopreservation/thawing method of neural stem cell balls and vitrification cryopreservation device

A neural stem cell and vitrification cryopreservation technology, which is applied in the field of neural stem cell sphere vitrification cryopreservation/resuscitation methods and cryopreservation devices, can solve the problems of difficult penetration of preservation agents, poor cell resuscitation activity, and large cell damage, and achieves low cost. , The effect of high mechanical strength and low temperature damage of cells

Active Publication Date: 2018-11-30
国源细胞工程有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problems of neural stem cell spheroids in the cryopreservation process, it is difficult for the preservation agent to penetrate into the interior of the cell spheroids, and the internal cell damage is large during the cooling process, the cell recovery activity is poor, and the recovery rate is low. The present invention provides a neural stem cell spheroid vitrification storage And recovery method, high cell recovery rate, good activity after recovery

Method used

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  • Vitrification cryopreservation/thawing method of neural stem cell balls and vitrification cryopreservation device
  • Vitrification cryopreservation/thawing method of neural stem cell balls and vitrification cryopreservation device
  • Vitrification cryopreservation/thawing method of neural stem cell balls and vitrification cryopreservation device

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 A cell spheroid collection tube used for vitrification of neural stem cell spheroids.

[0032] as attached figure 1 And attached figure 2The cell spheroid collection tube shown is a hollow cylindrical structure with both ends closed, including a tube body (1), a screw cap (2) and a gasket made of silica gel (3); the tube body (1) is tightly fitted double-layer tube with openings at both ends; the outer tube (5) of the double-layer tube is shorter than the inner tube (4), and both ends of the inner tube (4) have external spirals; the screw cap (2) has the same The outer helix of the inner tube (4) matches the inner helix, the outer diameter of the screw cap (2) is the same as that of the outer tube (5); the gasket (3) is set on the outer side of the inner tube (4) , the outer diameter of the gasket (3) is the same as the outer diameter of the outer tube (5). After the screw cap (2) is screwed tightly, the gasket (3) is compressed, and the cell spheroid coll...

Embodiment 2

[0034] Example 2 Vitrification storage and recovery of neural stem cell spheres.

[0035] 3.1 Preparation of medium

[0036] Neural stem cell complete medium (Neurobasal medium containing 1 times B27, 20ng / mL EGF, 20ng / ml bFGF) and DMEM / F12 medium containing 1 times HEPES buffer were obtained from the market; different configurations were made according to the following contents Medium:

[0037] Freezing solution 1 is DMEM / F12 medium containing 1 times HEPES buffer supplemented with 1.2% HES (hydroxyethyl starch), 10% DMSO and 10% EG (ethylene glycol);

[0038] Freezing solution 2 is DMEM / F12 medium containing 1 times HEPES buffer supplemented with 1.2% HES (hydroxyethyl starch), 20% DMSO, 20% EG (ethylene glycol) and 0.5mol / L sucrose;

[0039] Recovery medium 1 is DMEM / F12 containing 1 times HEPES buffer supplemented with 1.2% HES (hydroxyethyl starch) and 0.2mol / L sucrose;

[0040] Recovery medium 2 is DMEM / F12 containing 1 times HEPES buffer supplemented with 1.2% HES (h...

Embodiment 4

[0058] Example 4 Vitrified cryopreservation and post-thaw activity of neural stem cell spheres.

[0059] The same batch of neural stem cells as in Example 3 was cryopreserved and revived by the usual programmed cooling method, and revived after 6 months of cryopreservation.

[0060] Use the Trypan Blue method to detect the cell number and cell viability of the neural stem cell spheroids frozen in Example 3 and the common programmed cooling method before freezing and after recovery; and pass 5 times continuously, observe and calculate the cell passage cycle and value-added rate. From the data in Table 1-3, it can be seen that the average recovery rate of neural stem cell spheres after vitrification and recovery reaches more than 85%; and the cell subculture cycle and value-added rate are not significantly different from those before freezing; all data are better than the current programmed cooling method. cells after recovery.

[0061] Table 1 Recovery rate of different cryop...

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Abstract

The invention provides a cell ball collecting tube for vitrification cryopreservation of neural stem cell balls. The cell ball collecting tube is a hollow cylindrical structure with two closed ends, and comprises a tube body, rotary covers and gaskets; the tube body is a double-layer tube formed by tight sleeve, and two ends of the tube body are open; an outer tube of the double-layer tube is shorter than an inner tube; two ends of the inner tube are provided with an external spiral; each rotary cover is provided with an internal spiral which is in screw match with the corresponding external spiral of the inner pipe; the outer diameter of each rotary cover is the same as the outer diameter of the outer pipe; the gaskets sleeve the outer side of the inner pipe; and the outer diameter of thegasket is the same as the outer diameter of the outer pipe. The invention further provides a method for vitrification cryopreservation and thawing of neural stem cell balls by adopting the cell ballcollecting tube. Through the adoption of the method and a device for vitrification cryopreservation and thawing of neural stem cell balls, the vitrification cryopreservation of the neural stem cells can be efficiently completed.

Description

technical field [0001] The invention belongs to the field of cell preservation, and in particular relates to a method for vitrification / resuscitation of neural stem cell spheres and a freezing device. Background technique [0002] Cell cryopreservation is a technology that stores cells in a low temperature environment to reduce cell metabolism for long-term storage. Cell cryopreservation is one of the main methods of cell preservation. Using cryopreservation technology to store cells in liquid nitrogen at low temperature can make cells temporarily out of the growth state and preserve their cell characteristics, so that they can be recovered when needed. in the experiment. Moreover, moderately preserving a certain amount of cells can prevent the cells from being lost due to contamination of the cells being cultured or other accidents, which plays a role in cell preservation. [0003] Before the cells cultured in the laboratory are placed in liquid nitrogen or ultra-low temp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0242A01N1/0268
Inventor 雷云霆张国宁刘喆夏龙钢
Owner 国源细胞工程有限责任公司
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