A kind of neural stem cell sphere vitrification cryopreservation/resuscitation method and 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 preservatives, poor cell resuscitation activity, and large cell damage, and achieves low cost. , The effect of high mechanical strength and low temperature damage reduction of cells
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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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