COMPOSITION
- NK Cell Initial Flask
- NK Cell Initial Medium
- NK Cell Initial Cocktail
- NK Cell Subculture Medium
- [Other supplies required]
- Ficoll-Paque (GE Healthcare, Sweden)
- Sterile PBS
- FBS or autologous plasma (It is desirable to be heat-inactivated at 56°C for 30 minutes.)
- Sterile conical centrifuge tubes
Natural killer (NK) cells can be expanded from human peripheral blood mononuclear cells (PBMCs) without using feeder cells.
NK cells can be expanded several hundred to several thousand-fold by 2 - 3 weeks of culture.
One kit issufficient toexpand NK cells from 20 - 50 ml of whole blood.
Procedures
- Preparing reagentsNK Cell Initial Medium and NK Cell Subculture Medium should be supplemented with 5 % (v/v) of heat-inactivated FBS or autologous plasma.
- Preparing peripheral blood mononuclear cells (PBMCs)
- 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)
- Sterile PBS
- FBS or autologous plasma (It is desirable to have them heat-inactivated at 56 ℃ for 30 minutes.)
- Sterile conical centrifuge tubes
- Sterile culture flasks
Precautions
- 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.
References
- 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
- 鄧学文、芦葉恵介、照沼篤、高根翼、鮫島葉月、贄田美江、照沼裕: 培養NK細胞の投与による体内NK細胞の活性化. *Medical
Science Digest* 41, 42-45, 2015.
- 鄧学文、芦葉恵介、照沼篤、高根翼、鮫島葉月、贄田美江、照沼裕: 培養NK細胞の投与による体内NK細胞の活性化. *Medical
Science Digest* 41, 42-45, 2015.
- 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
