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
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[0029] Example 1 Cell spheroid collection sheet for vitrification and cryopreservation of neural stem cell spheroids.
[0030] as attached figure 1 and attached figure 2 The cell spheroid collection sheet shown includes an adherent surface (1) and a support (2); the adherent surface (1) is a rectangle of 20mm×3.5mm; the support (2) and the short side of the adherent surface (1) They are connected vertically and are L-shaped; 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 γ-ray irradiation after TC treatment.
[0031] Pre-coated cell pellets: first use 0.01% poly-ornithine at 0.1 mL / cm 2 Coat, 37℃, 1h. Wash twice with PBS, then use 10mg / L laminin solution at 1.5μg / cm 2 Coat, 37℃, 2h, wash twice with PBS.
Example Embodiment
[0032] Example 2 Vitrification and recovery of neural stem cell spheroids.
[0033] 2.1 Preparation of culture medium
[0034] Neural stem cell complete medium (Neurobasal medium containing 1x B27, 20ng / mL EGF, 20ng / ml bFGF) and DMEM / F12 (containing HEPES buffer) medium were purchased from the market; different cultures were prepared according to the following contents. base:
[0035] Freezing solution 1 is DMEM / F12 medium containing 1x 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 1x 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 1x HEPES buffer supplemented with 1.2% HES (hydroxyethyl starch) and 0.2mol / L sucrose;
[0038] Recovery medium 2 is DMEM / F12 containing 1x HEPES buffer supplemented with 1.2% HES (hydroxyethyl starch) and 0.1mol / L...
Example Embodiment
[0055] Example 3 Neural stem cell spheroid vitrification and post-recovery activity.
[0056] The neural stem cells of the same batch as in Example 3 were frozen and thawed by a common procedure cooling method, and thawed after 6 months of freezing.
[0057] The trypan blue method was used to detect the cell number and cell viability of the neural stem cell spheres frozen in Example 3 and the commonly used programmed cooling method before cryopreservation and after resuscitation; and serially passaged 5 times to observe and calculate the cell passage cycle and appreciation rate. From the data in Table 1-3, it can be seen that the average recovery rate of neural stem cell spheres after vitrification cryopreservation and recovery reaches more than 85%; and the cell passage cycle and proliferation rate are not significantly different from those before cryopreservation; all data are better than the current programmed cooling method cryopreservation post-recovery cells.
[0058] ...
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