METHOD FOR PRODUCING NATURAL KILLER CELLS
The present disclosure relates to a method for producing natural killer (NK) cells. More specifically, the present disclosure relates to a method for producing NK cells, characterized in that peripheral blood mononuclear cells from which CD3-positive cells are removed are proliferated together with feeder cells, and the peripheral blood mononuclear cells are re-stimulated with feeder cells at the time of reaching a specific accumulated population doubling level. The present disclosure also relates to a method for producing NK cells, characterized in that NK cells are cultured under appropriate culture conditions by using a bioreactor. The production method according to the present disclosure has an advantage that NK cells having a high cell-killing ability and cell survival rate can be produced with high purity and at high efficiency in a short period of time by a clinically friendly method as compared with existing methods, thereby increasing the productivity of an NK cell therapy agent.
1 . A method for producing NK cells, comprising:
(a) stimulating a cell culture comprising NK cells with feeder cells and then culturing the same stationarily;
(b) suspension-culturing the stationarily cultured cell culture; and
(c) re-stimulating the suspension-cultured cell culture by adding feeder cells at the time when the accumulated population doubling level of mononuclear cells in the cell culture reaches 3-5 and then suspension-culturing the same.
2 . The method for producing NK cells according to claim 1 , wherein the suspension culturing of the step (b) is initiated 3-7 days after the stimulation with feeder cells in the step (a).
3 . The method for producing NK cells according to claim 1 , wherein the suspension culturing of the step (c) is performed at an agitation speed of 30-300 rpm.
4 . The method for producing NK cells according to claim 1 , wherein the culturing of the step (a) is performed in a medium to which an anti-CD3 antibody is added.
5 . The method for producing NK cells according to claim 4 , wherein the anti-CD3 antibody is one or more selected from a group consisting of OKT3, UCHT1 and HIT3a.
6 . The method for producing NK cells according to claim 1 , wherein the culturing of the step (a) is performed in a medium to which one or more cytokine selected from a group consisting of interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18) and interleukin-21 (IL-21) is added.
7 . The method for producing NK cells according to claim 1 , wherein the feeder cells are inactivated peripheral blood mononuclear cells.
8 . The method for producing NK cells according to claim 1 , wherein the cell culture of the step (a) comprises peripheral blood mononuclear cells from which CD3-positive cells are removed.
9 . A method for producing NK cells, comprising a step of inoculating a cell culture comprising NK cells to a bioreactor.
10 . The method for producing NK cells according to claim 9 , wherein the bioreactor is controlled to pH 6.5-7.6, culture temperature of 25-40° C. and agitation power per unit volume of 0.1-100 W/m 3 .
11 . The method for producing NK cells according to claim 9 , wherein the cell culture is subjected to stationary culture and suspension culture sequentially.
12 . The method for producing NK cells according to claim 9 , wherein the concentration of NK cells in the bioreactor at the time of the inoculation is 0.1-2.0×10 6 cells/mL.
13 . The method for producing NK cells according to claim 9 , wherein a target cell concentration (cell concentration after addition of additives, or feeding target cell density) during the culture period is 0.4-1.0×10 6 cells/mL.
14 . The method for producing NK cells according to claim 9 , wherein the cell culture is a culture of the NK cells produced by the method according to any of claims 1 to 8 .
15 . NK cells produced by the method according to claim 1 .
16 . NK cells produced by the method according to claim 9 .