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A technology of cells and resuscitation fluid, applied in the field of CAR-T cells, to achieve good survival rate, facilitate industrial use, and optimize the composition of resuscitation fluid
Active Publication Date: 2020-12-18
广东康盾高新技术产业集团股份公司
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However, during the research process, it was found that after using this cryopreservation solution, if the conventional resuscitation method is used, the cryopreserved cells still cannot achieve the best proliferation ability and the ability to remove tumor cells.
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Embodiment 1
[0023] A recovery method for CAR-T cells, comprising the following steps:
[0024] Take out the cryopreservation bag containing CAR-T cells and cryopreservation solution from liquid nitrogen, add resuscitation solution preheated at 36-40°C, shake the cryopreservation bag in a water bath at 36-40°C for 1-2 minutes, and centrifuge at 500rpm for 1 ~2min, discard the supernatant; add resuscitation solution preheated at 36~40℃, centrifuge at 500rpm for 1~2min, discard the supernatant, and resuspend the cells with RPMI-1640 medium; 2 to 3 times the volume of the freezing solution.
[0025] The resuscitation solution includes the following components: L-proline 50 μg / L, sodium selenite 10 mg / L, sodium pyruvate 2.2 mg / L, NaOH 0.2 mg / L, magnesium chloride 0.2 mg / L, astragaloside 4 25 mg / L L, Atractylodes lactone II 10mg / L, sodium diatrizoate 30mg / L, and the balance is RPMI-1640 medium.
Embodiment 2
[0027] A recovery method for CAR-T cells, comprising the following steps:
[0028] Take out the cryopreservation bag containing CAR-T cells and cryopreservation solution from liquid nitrogen, add resuscitation solution preheated at 36-40°C, shake the cryopreservation bag in a water bath at 36-40°C for 1-2 minutes, and centrifuge at 600rpm for 1 ~2min, discard the supernatant; add resuscitation solution preheated at 36~40℃, centrifuge at 600rpm for 1~2min, discard the supernatant, and resuspend the cells with RPMI-1640 medium; 2 to 3 times the volume of the freezing solution.
[0029] The resuscitation solution includes the following components: L-proline 50 μg / L, sodium selenite 10 mg / L, sodium pyruvate 2.2 mg / L, NaOH 0.2 mg / L, magnesium chloride 0.2 mg / L, astragaloside 4 25 mg / L L, Atractylodes lactone II 10mg / L, sodium diatrizoate 30mg / L, and the balance is RPMI-1640 medium.
Embodiment 3
[0031] The difference between this example and embodiment 1 is:
[0032] The resuscitation fluid includes the following components: L-proline 25 μg / L, sodium selenite 50 mg / L, sodium pyruvate 2.2 mg / L, NaOH 0.5 mg / L, magnesium chloride 0.4 mg / L, astragaloside IV 20 mg / L L, Atractylodes Ⅱ 25mg / L, sodium diatrizoate 18mg / L, and the balance is RPMI-1640 medium.
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Abstract
The invention discloses a resuscitation method of CAR-T cells. The resuscitation method comprises the following steps: taking out a cryopreservation bag loaded with CAR-T cells and cryopreservation liquid from liquid nitrogen, adding cryopreservation liquid preheated at 36-40 DEG C, shaking the cryopreservation bag for thawing under the condition of 36-40 DEG C water bath for 1-2 minutes, performing centrifuging, and discarding supernate; and adding resuscitation liquid preheated at 36-40 DEG C, performing mixing, performing centrifuging, discarding supernate, and resuspending the cells by using a cell culture medium, wherein the resuscitation liquid comprises the following components of 10-50 [mu] g/L L-proline, 10-100 mg/L sodium selenite, 2.0-2.2 mg/L sodium pyruvate, 0.2-0.5 mg/L f NaOH, 0.2-0.4 mg/L magnesium chloride, 15-25 mg/L astragaloside, 10-50 mg/L atractylodes macrocephala lactone II, 15-30 mg/L sodium diatrizoate and the balance the cell culture medium. By adopting the resuscitation method disclosed by the invention, the motility rate, the multiplication capacity and tumor cell removal capacity of cells can be well maintained. And the method is simple and convenient to operate, low in cost and beneficial to industrial use.
Description
technical field [0001] The present invention relates to the technical field of CAR-T cells, in particular to a recovery method for CAR-T cells. Background technique [0002] CAR-T cells are a modified T cell obtained by introducing an artificially designed exogenous chimeric antigen receptor (CAR) into T cells. CAR-T cells have attracted much attention due to their good therapeutic effects in the treatment of leukemia, lymphoma, melanoma, glioma and other malignant tumors. During the application of CAR-T cells, due to the influence of factors such as the preparation cycle of cell preparations and the timing of treatment, it is often necessary to freeze them in liquid nitrogen and resuscitate them before use. When resuscitating cryopreserved cells, usually put the cryopreservation tube or cryopreservation bag directly at 37°C to thaw quickly, then add complete growth medium to resuspend the cells, discard the supernatant after centrifugation, and add medium to continue cultu...
Claims
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