Medium composition for cryopreservation of cell and use thereof
A kind of technology of composition and culture medium, applied in the field of pharmaceutical composition of therapeutic cells
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Embodiment 1-3
[0062] Example 1-3 Preparation of medium composition for cryopreservation
[0063] To prepare a medium composition for cryopreservation, 5 ml of DMSO, 25 ml of dextran 40, 50 ml of albumin injection, and 20 ml of RPMI1640 as a cell culture medium were mixed (Example 1). In Examples 2 and 3, the same method as in Example 1 was also used but the amount shown in Table 1 below was used to prepare a freezing medium composition containing dimethyl sulfoxide (DMSO, Bioniche Pharma, USA) , Dextran 40 (Dai Han Pharm. Co., Ltd), albumin injection (Korea Green Cross), and RPMI1640 (Gibco, USA).
experiment example 1
[0069] Experimental Example 1 Evaluation of NK cells after freezing and thawing
[0070] 1.1 Freezing and thawing of NK cells
[0071] NK cells were frozen in the medium composition for freezing storage prepared in Example 1-3 and Comparative Example 1-6, and then thawed to evaluate the medium composition for freezing storage.
[0072] First, monocytes were isolated from human peripheral blood (Seoul National University Hospital, South Korea). The CliniMACS system was used to remove CD3 positive cells from the isolated monocytes and use them as seed cells. At the same time, monocytes irradiated with 2000cGy of gamma rays were used as support cells for NK cell culture. Divide seed cells and supporting cells to 0.5 x 10 6 Up to 1 x10 6 The concentration of cells / ml is cultured at SCGM medium (CellGenix, Germany), the medium is supplemented with 500IU / ml IL-2, 10μg / ml anti-CD3 antibody OKT3 and 1v / v% serum. At 37°C and 5% CO 2 Cultivate for 14-21 days under the same conditions.
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experiment example 2
[0112] Experimental example 2 Evaluation of tumor cells after thawing
[0113] It was checked whether the medium composition for freezing storage prepared in Examples 1-3 and Comparative Examples 1-6 can also be used to freeze tumor cells.
[0114] First, at 37°C and 5% CO 2 The human T lymphoma cell line HuT78 (ATCC, United States) and human leukemia cell K562 (ATCC, United States) were cultured in RPMI1640 medium containing 10% fetal bovine serum and RPMI1640 medium containing 20% fetal bovine serum under the conditions of ). The cultured cells were recovered and suspended in each medium. Then, place HuT78 cells and K562 cells in the vial, and place them in a volume of 1 x 10 7 Cells / ml and 4 x 10 6 The concentration of cells / ml is frozen. Cell freezing was performed by the same method as described in Experimental Example 1.1 above. Resuspend the frozen cells in each medium, and subculture the cells to obtain 1 x 10 6 Concentration of cells / ml at 37°C and 5% CO 2 Cultivate f...
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