US 12,376,584 B2
In-vitro culture, induction, activation and cryopreservation method and cell bank establishment for immune cells
Bingqiang Zhang, Qingdao (CN); and Mengmeng Chen, Qingdao (CN)
Appl. No. 17/634,556
Filed by Qingdao Restore Biotechnology Co., Ltd., Qingdao (CN)
PCT Filed May 26, 2020, PCT No. PCT/CN2020/092287
§ 371(c)(1), (2) Date Feb. 11, 2022,
PCT Pub. No. WO2021/223274, PCT Pub. Date Nov. 11, 2021.
Claims priority of application No. 202010371435.2 (CN), filed on May 6, 2020.
Prior Publication US 2023/0051425 A1, Feb. 16, 2023
Int. Cl. A01N 1/125 (2025.01); A01N 1/162 (2025.01); C12N 5/0783 (2010.01); C12N 15/10 (2006.01)
CPC A01N 1/125 (2025.01) [A01N 1/162 (2025.01); C12N 5/0646 (2013.01); C12N 15/1037 (2013.01); C12N 2500/90 (2013.01); C12N 2501/115 (2013.01); C12N 2501/117 (2013.01); C12N 2501/14 (2013.01); C12N 2501/2301 (2013.01); C12N 2501/2302 (2013.01); C12N 2501/231 (2013.01); C12N 2501/235 (2013.01); C12N 2501/37 (2013.01); C12N 2501/998 (2013.01); C12N 2533/52 (2013.01)] 4 Claims
 
1. An in-vitro culture, induction, activation and cryopreservation method and cell bank establishment for immune cells, comprising the followings: using a dedicated amplification medium of immune cells to perform first-stage amplification culture on mononuclear cells to obtain preliminarily amplified immune cells; wherein the dedicated amplification medium of immune cells is a serum-free lymphocyte medium added with 500-2000 IU/ml IL-2, 500-2000 IU/ml IL-10, 0.5-1 ng/ml IL-1α and 1-4 ng/ml LIF, 0.5-2.5 ng/ml EPO, 1-4 ng/ml KGF, 2-5 ng/ml testosterone, 1-4 μg/ml parathyroid hormone and 1-4 μg/ml laminin; using a dedicated induction medium of immune cells to perform second-stage induction and amplification culture on the preliminarily amplified immune cells to obtain induced immune cells; using a dedicated activation medium of immune cells to perform third-stage activation and amplification culture on the induced immune cells to obtain a large number of immune cells with activation functions; using a dedicated cryopreserving fluid of immune cells to cryopreserve the immune cells to obtain cryopreserved immune cells; and performing preservation according to ABO/RH typing and HLA typing; and establishing an information file of immune cells for retrieval to construct an immune cell bank; wherein the immune cells are natural killer cells.