PBMCs should be isolated from the whole blood by density gradient centrifugation using Ficoll-Paque.
・Washing an NK cell Initial Flask
10 ml PBS should be added to an NK Cell Initial Flask. Slant the flask to cover the entire surface with PBS. Aspirate the liquid completely from the flask. Care should be taken not to scratch the inner surface of the flask. Repeat the washing process two more times.
・Culturing NK cells from PBMCs
PBMCs should be suspended in the NK Cell Initial Medium at 1 x 106 cells/ml. Add 40 μl of NK Cell Initial Cocktail to the 1 ml of the cell suspension. Seed the cells onto the pre-washed NK Cell Initial Flask, and incubate under 5 % CO2 at 37 ℃ for 3 days.
・Changing medium and sub-culturing
Transfer floating as well as adherent cells to conical centrifuge tube and centrifuge at 200 x g for 8 minutes. Remove the supernatant and re-suspended the cells at 1 x 106 cells/ml in the NK Cell Subculture Medium supplemented with 5 % (v/v) of heat- inactivated FBS or autologous plasma. The cell suspension is transferred to conventional culture flasks and incubated under 5 % CO2 at 37 ℃. Cells should be sub-cultured every 2 - 3 days by suspending cells in the completed NK Cell Subculture Medium at 0.8 x 106 cells/ml.
・Suggested culturing period: 2 to 3 weeks.
Other supplies required
Ficoll-Paque (GE Healthcare, Sweden)
FBS or autologous plasma (It is desirable to have them heat-inactivated at 56 ℃ for 30 minutes.)
Sterile conical centrifuge tubes
Sterile culture flasks
NK Cell Initial Flask may carry condensation on the surface, which does not adversely affect the performance of the kit. This product contains human plasma albumin.
Chu Thi Thao, Do Thi Hoai Thu, Bui Viet Anh, Truong Linh Huyen, Nguyen Van Phong, Nguyen Thanh Liem, and Hoang Thi My Nhung: Efficient Expansion of Human Umbilical Cord Blood- Derived NK Cells Ex Vivo without Requiring Feeder Layers, Biomed J Sci & Tech Res, 8: 6739-6744 DOI: 10.26717/BJSTR.2018.08.001711
Hoang Thi My Nhung, Bui Viet Anh, Truong Linh Huyen, Doan Trung Hiep, Chu Thi Thao, Phung Nam Lam, Nguyen Thanh Liem: Ex vivo expansion of human peripheral blood natural killer cells and cytotoxic T lymphocytes from lung cancer patients, Oncol Lett, 15: 5730-5738, 2018
Deng X, Terunuma H, Terunuma A, Takane T, Nieda M: Ex vivo-expanded natural killer cells kill cancer cells more effectively than ex vivo-expanded gd T cells or ab T cells. Int Immunopharmacol 22: 486-491, 2014
Terunuma H, Deng X, Nishino N, Watanabe K: NK cell-based autologous immune enhancement therapy (AIET) for cancer. Stem Cells Regenerative Med 9: 9-13, 2013
Deng X, Terunuma H, Nieda M, Xiao W, Nicol A: Synergistic cytotoxicity of ex vivo expanded naturel killer cells in combination with monoclonal antibody drugs against cancer cells. Int Immunopharmacol 14: 593-605, 2012
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