Peripheral blood mononuclear cell serum-free freezing medium and freezing method
A cryopreservation method and nuclear cell technology, applied in the field of serum-free cryopreservation and cryopreservation of peripheral blood mononuclear cells, can solve the problems that the cell culture medium cannot be directly injected into the human body, affect the effect of cell therapy, and damage the respiratory system, etc. Achieve the effect of avoiding the risk of spreading potential pathogens, improving safety and reducing damage
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Embodiment 1
[0032] A method for isolating human peripheral blood mononuclear cells, comprising the following steps:
[0033] 1. Isolation of PBMCs
[0034] 1.1 Use a 10mL disposable pipette to transfer 30ml of peripheral blood added with sodium citrate anticoagulant to a 50mL centrifuge tube, and centrifuge at 800g for 10min.
[0035] 1.2 After centrifugation, use a Pasteur pipette to suck off the upper layer of plasma, use a 10mL disposable pipette to add 20ml of normal saline to dilute, and mix well.
[0036] 1.3 Take another new 50mL centrifuge tube, add 12mL lymphocyte separation solution (blood dilution solution: lymphatic separation solution = 2:1) into each tube with a 10mL disposable pipette, and use a 10mL disposable pipette to dilute the The blood slowly transfers to the surface of the lymphocyte separation medium, so that a clear interface is formed between the two.
[0037] 1.4 Centrifuge at 700g for 30min, and change the centrifuge lift rate to 0.
[0038] 1.5 After centri...
Embodiment 2
[0043] The serum-free cryopreservation solution for human peripheral blood mononuclear cells is prepared by mixing 5-20% dimethyl sulfoxide, 0.5-10% dextran 40 and the balance Bomali A by volume percentage.
[0044] Component A of Bomaili is: every 1000ml contains 5.26g of sodium chloride, 5.02g of sodium gluconate, 3.68g of sodium acetate, 0.37g of potassium chloride, and 0.30g of magnesium chloride.
[0045] In a further specific scheme, the above-mentioned serum-free cryopreservation solution for human peripheral blood mononuclear cells can be prepared by volume percentage, containing 20% DMSO, 2% dextran 40, and the balance being Bomalil A. The above serum-free cryopreservation solution for human peripheral blood mononuclear cells can also be prepared by volume percentage, containing 10% DMSO, 1% dextran 40, and the balance being Bomali A.
Embodiment 3
[0047] The serum-free cryopreservation method of human peripheral blood mononuclear cells, the specific steps are:
[0048] 1. Take 10-20 ml of the cell cryopreservation solution prepared in Example 2 (20% DMSO+2% dextran 40+Bomalis A).
[0049] 2. Take the PBMC cell pellet obtained by separating in Example 1.
[0050] 4. Add Bomalis A to the PBMC cell pellet to make the cell density 1~2×10 7 / ml, mix by gently pipetting.
[0051] 5. Slowly add the cell freezing solution prepared in step 1 equal to the volume of the cell suspension in the tube to the above PBMC cell suspension along the tube wall, and gently blow and mix to make the cell density (0.5~1)×10 7 / ml.
[0052] 6. Divide the cell suspension into cryopreservation tubes, 1ml per tube.
[0053] 7. Make a mark on the cryopreservation tube, including the umbilical cord number, cell generation number, cell cryopreservation batch number, date of cryopreservation, operator and other relevant information.
[0054] 8. Pl...
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