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Neural stem cell sphere vitrified cryopreservation/resuscitation method

A neural stem cell, vitrification cryopreservation technology, applied in the field of vitrification cryopreservation/resuscitation of neural stem cell spheres, can solve the problems of difficult penetration of cryopreservatives and large cell damage, and achieve efficient vitrification cryopreservation, reduction of cell low temperature damage, and improvement of The effect of penetration efficiency

Active Publication Date: 2018-07-20
国源生命科学集团有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the problems that the cryopreservation agent is difficult to penetrate into the interior of the cell ball during the cryopreservation process of the neural stem cell ball, and the internal cell damage is large during the cooling process, the present invention provides a vitrification cryopreservation and recovery method of the neural stem cell ball, and the cell recovery rate is high. , good activity after recovery

Method used

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  • Neural stem cell sphere vitrified cryopreservation/resuscitation method
  • Neural stem cell sphere vitrified cryopreservation/resuscitation method
  • Neural stem cell sphere vitrified cryopreservation/resuscitation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 A collection sheet of cell spheres used for vitrification of neural stem cell spheres.

[0030] as attached figure 1 And attached figure 2 The cell spheroid collection sheet shown includes the adherent surface (1) and the bracket (2); the adherent surface (1) is a rectangle of 20mm×3.5mm; the short side of the bracket (2) and the adherent surface (1) connected vertically in an L shape; the height of the bracket (2) is less than the length of the wall-adhering surface (1), so that the wall-adhering surface (1) forms an angle of 15° with the horizontal plane. The cell spheroid collection sheet is made of polystyrene, and the surface is sterilized by gamma-ray irradiation after TC treatment.

[0031] The cell spheroid collection sheet is coated in advance: first use 0.01% poly-ornithine at 0.1mL / cm 2 Coating, 37°C, 1h. Wash twice with PBS, then use 10mg / L laminin solution to press 1.5μg / cm 2 Coating, 37 ° C, 2h, PBS washed twice.

Embodiment 2

[0032] Example 2 Vitrification storage and recovery of neural stem cell spheres.

[0033] 2.1 Preparation of medium

[0034] Neural stem cell complete medium (Neurobasal medium containing 1 times B27, 20ng / mL EGF, 20ng / ml bFGF) and DMEM / F12 (containing HEPES buffer) medium were purchased from the market; different cultures were configured according to the following contents base:

[0035] 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);

[0036]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;

[0037] Recovery medium 1 is DMEM / F12 containing 1 times HEPES buffer supplemented with 1.2% HES (hydroxyethyl starch) and 0.2mol / L sucrose;

[0038] Recovery medium 2 is DMEM / F12 containing 1 times HEPES buffer supplemented with 1.2% HES (hydroxyet...

Embodiment 3

[0055] Example 3 Vitrification storage and post-thaw activity of neural stem cell spheres.

[0056] 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.

[0057] 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.

[0058] Table 1 Recovery rate of different cryopreser...

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Abstract

The invention provides a neural stem cell sphere vitrified cryopreservation and resuscitation method. The method comprises the steps of enabling neural stem cell spheres to be attached to a cell sphere collection piece; then, incubating the cell sphere collection piece in a cryopreservation solution, and then freezing in liquid nitrogen; then, transferring to a cryopreservation tube for storage inliquid nitrogen; during resuscitation, incubating the cell sphere collection piece in a resuscitation medium for resuscitation, culturing in a complete medium of neural stem cells and then subcultring, and culturing and subcultring in a normal manner; the cell sphere collection piece comprises a wall-adherent surface and a support, wherein the wall-adherent surface is rectangular; the support isperpendicularly connected with the short side of the wall-adherent surface and is in an L shape; the height of the support is less than the length of the wall-adherent surface. The neural stem cell sphere vitrified cryopreservation and resuscitation method provided by the invention can bind the neural stem cell spheres to the smaller wall-adherent surface in a high density, thus reaching the precondition of vitrified cryopreservation; furthermore, the cell spheres are attached to the wall in a spreading way, so that the thickness of the cell spheres is effectively reduced, and the penetrationefficiency of a cryoprotectant is increased.

Description

technical field [0001] The invention belongs to the field of cell preservation, and in particular relates to a vitrification preservation / resuscitation method of neural stem cell spheres. 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 temperature refri...

Claims

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Application Information

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IPC IPC(8): A01N1/02C12N5/0797
CPCA01N1/0221C12N5/0623C12N2533/12C12N2533/30
Inventor 雷云霆张国宁刘喆夏龙钢
Owner 国源生命科学集团有限公司
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