Preparing method of nerve cell cryoprotectant
A neuron and cryopreservation technology, which is applied in the field of neuron cryopreservation preparation, can solve the problems of low neuron cryopreservation and resuscitation rate, and achieves a process that is beneficial to digestion, reduces cryopreservation damage, and improves sampling efficiency. Effect
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[0031] The present invention also provides a method for preparing a neuron cryopreservation solution, comprising the following steps:
[0032] Sampling: Take the isolated mammalian nervous tissue, shred it and place it in the first centrifuge tube for 4-6 minutes at 800-1200rpm, and remove the centrifuged supernatant;
[0033] Digestion: Add trypsin to the first centrifuge tube, blow off the precipitated cells in the first centrifuge tube, draw out the precipitated cells and place them in the digestion culture dish, add trypsin to the digestion culture dish, place the digestion culture dish at 37 After 8-12 minutes in the incubator at ℃, add the planting medium to the digestion culture dish to terminate the digestion;
[0034] Preparation of cell suspension: Filter the mixture in the digestion dish through a cell sieve, centrifuge at 800-1200rpm for 4-6min, remove the supernatant after centrifugation, and obtain primary neurons;
[0035] Cryopreservation: mix the primary neur...
Embodiment 1
[0058] (1) Experimental preparation: Coat four 24-well plates and two 6-well plates with poly-lysine, remove the coating solution after staying overnight at 37°C, blow it for 2 hours until dry; wash with sterile water twice, add sugar Type DMEM medium 0.3mL / well, remove the DMEM medium before cell planting, replace the 24-well plate with 500μL planting medium, and replace the 6-well plate with 2mL planting medium; sterilize surgical instruments, including scissors, tweezers and fiber tweezers .
[0059] (3) Disinfect the microscope, and anesthetize the animal to be tested in this implementation—the 5-day-old embryonic mouse.
[0060] (4) Sampling: Cut open the skull of the embryonic mouse, take out the brain tissue, peel off the blood vessels and meninges in ice-cold DMEM medium, cut the brain tissue into pieces, take out the brain tissue, put it in a 15mL first centrifuge tube and centrifuge at 1000rpm for 5min, remove Centrifuge the supernatant.
[0061] (5) Digestion: Add...
Embodiment 2
[0073] The difference between Example 2 and Example 1 is that in the recovery step, the frozen neurons are transferred to the recovery medium for culture. The proportioning ratio of the planting medium, neuron medium, neural stem cell medium, cryopreservation solution, and recovery medium used in Example 2 is as follows:
[0074] Volume ratio of planting medium components: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL double antibody.
[0075] Volume ratio of neuron medium components: 50.00mL Neurobasal A, 1.00mL B27, 0.30mL double antibody.
[0076] Volume ratio of neural stem cell medium components: 45.00mL DMEM / F12, 1.00mLbFGF, 1.00mLEGF, 1.00mLB27, 0.50mLN2, 0.50mL double antibody.
[0077] The volume ratio of the cryopreservation solution components: 50.00mL neuron culture medium, 45.00mL 10% bovine serum albumin, 5.00mL dimethyl sulfoxide.
[0078] Volume ratio of recovery medium components: 50.00mL neuron medium, 50.00mL neural stem cell medium, 1.0mL 10% BSA and 1.0mL ...
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