METHOD FOR PREPARING CHIMERIC ANTIGEN RECEPTOR T CELLS BY SERUM-FREE CULTURE
Provided is a method of preparing chimeric antigen receptor T cells by serum-free culture. The method comprises steps of: (a) providing PBMC cells; (b) performing negative sorting treatment on the PBMC cells to obtain sorted PBMC cells; (c) activating the sorted PBMC cells to obtain activated T cells; (d) transfecting the activated T cells with a viral vector expressing a chimeric antigen receptor, so as to obtain transfected T cells; (e) removing the viruses from the transfected T cells to obtain virus-removed T cells; and (f) subjecting the virus-removed T cells to expansion culture to obtain chimeric antigen receptor T cells.
1 . A serum-free method of preparing chimeric antigen receptor T cells, wherein the method comprises steps of:
(a) providing PBMC cells, which are resuspended in a serum-free medium;
(b) performing negative sorting treatment on the PBMC cells at step (a) to obtain sorted PBMC cells;
(c) activating the sorted PBMC cells obtained at step (b) to obtain activated T cells;
(d) performing gene transfection on the activated T cells obtained at step (c) with a viral vector expressing a chimeric antigen receptor to obtain transfected T cells;
(e) removing the viruses from the transfected T cells to obtain virus-removed T cells; and
(f) resuspending the virus-removed T cells in an expansion medium and performing expansion culture to obtain chimeric antigen receptor T cells, and harvesting the chimeric antigen receptor T cells when the chimeric antigen receptor T cells reach a predetermined quantity, wherein the expansion medium is a serum-free medium containing a cell factor.
2 . The method according to claim 1 , wherein a serum-free medium is used at all steps.
3 . The method according to claim 1 , wherein at step (c), the activation treatment is performed in an activation medium, which contains a basic medium and an additive.
4 . The method according to claim 1 , wherein the basic medium is selected from the following group: LONZA X-VIVO, LIFE CTS AIM V, LIFE OpTmizer SFM, RPMI 1640, ImmunoCult™-XF and Stemlineä.
5 . The method according to claim 1 , wherein the additive is selected from the following group: human serum albumin, recombinant human serum albumin, plant-derived recombinant albumin or a combination thereof, preferably recombinant albumin.
6 . The method according to claim 1 , wherein the expansion medium at step (f) further contains a cell factor
7 . The method according to claim 1 , wherein the cell factor is selected from the following group: IL-2, IL-15, IL-7 or a combination thereof.
8 . The method according to claim 1 , wherein at step (f), the expansion culture is performed in a container, which is selected from the following group: a culture flask, a culture bag, or a combination thereof.
9 . Chimeric antigen receptor T cells, wherein the chimeric antigen receptor T cells are prepared by the serum-free method of preparing chimeric antigen receptor T cells as described in claim 1 .
10 . A cell preparation, wherein the cell preparation contains (a) the chimeric antigen receptor T cells in claim 2 and (b) a pharmaceutically acceptable carrier.