OptiVitro NK Cell Culture System P01, Serum-free
Notice: This item will be shipped in multiple packages. Typically 1Box DRY ICE (Supplements/Proteins/Factors) and 1Box BLUE ICE PACK (Cell Culture Media and Others).
To ensure optimal storage conditions, some packages will contain dry ice and blue ice.
To ensure optimal shipping conditions, extra shipping charges may apply.
Cat. No.: EC-NE000-N022
Desciption
OptiVitro® NK Cell Expansion Serum-free Kit P01 has been specifically designed for the in vitro expansion of human Natural Killer (NK) cells derived from either peripheral blood mononuclear cells (PBMCs) or cord blood mononuclear cells (CB-MNCs). The kit is composed of five components: OptiVitro® NK Cell Basal SF Medium P01, OptiVitro® NK Cell SF Medium Supplement, OptiVitro® Cytokine I, OptiVitro® Cytokine II, and OptiVitro® Cytokine III. All of these components are serum-free, xeno-free, and have been manufactured in strict compliance with GMP regulations.
Instructions for use
Prepare media
1. Place OptiVitro® NK Cell Basal SF Medium P01 and OptiVitro® NK Cell SF Medium Supplement under a sterile laminar flow hood.
2. Add 4 mL/8 mL OptiVitro® NK Cell SF Medium Supplement to 500 mL/1000 mL OptiVitro® NK Cell Basal SF Medium P01.
3. Tighten and mix the complete OptiVitro® NK Cell Basal SF Medium P01 thoroughly.
Note：It is recommended to use complete OptiVitro® NK Cell Basal SF Medium P01 within four weeks after mixed.
4. To prepare the complete medium (short name: "NK-SFM"), add 155 µL/310 µL of OptiVitro® Cytokine III to 500 mL/1000 mL of the previously prepared OptiVitro® NK Cell Expansion Serum-free Medium P01.
Note:
1) The complete medium (the short name is ‘NK-SFM’ in the following protocol) is stable for 3 weeks when stored at 2-8℃ in the dark.
2) OptiVitro® Cytokine III can be aliquoted for small volume culture use but should be limited to three freezethaw cycles.
Culture NK cells from PBMCs
OptiVitro® NK Cell Expansion Serum-free Kit P01 is designed for culturing NK cells from peripheral blood mononuclear cells (PBMCs), cord blood mononuclear cells (CB-MNCs), or NK cells derived from iPS cells. The kit does not necessarily require serum or serum replacement, but supplementing with heat-inactivated autologous plasma, serum replacement, or human AB serum can increase cell expansion folds. This protocol outlines the procedures for culturing NK cells from PBMCs, starting with a T75 flask as an example.
1. Thaw OptiVitro® Cytokine I at room temperature one day before NK cell activation. Add 45 µL OptiVitro® Cytokine I to 15 mL sterile DPBS and mix well. Transfer the mixed liquid to a sterile T75 flask, shaking it slightly to ensure the liquid covers the bottom of the flask. Store the flask at 2-8℃ overnight.
2. Prepare fresh PBMCs following standard PBMC separation protocols or quickly thaw (<1 minute) frozen vials of PBMC cells in a 37°C water bath.
3. If using fresh PBMCs, wash them with sterile DPBS and use them directly. If using frozen cells, thaw them one day before NK cell activation, place them at a concentration of around 2×106 cells/mL in complete OptiVitro® NK Cell Expansion Serum-free Medium P01 without extra cytokines, and incubate them in a humidified 37℃ incubator with an atmosphere of 5% CO2 for 16-24 h.
4. It is optional to sort NK cells using magnetic beads with antibodies before the activation.
5. Centrifuge cells at 400×g for 10 minutes and discard the supernatant.
6. Equilibrate the T75 flask coated with OptiVitro® Cytokine I (prepared in step 1) at room temperature and remove the liquid.
7. Equilibrate complete OptiVitro® NK Cell Expansion Serum-free Medium P01 to room temperature before use. Resuspend the PBMCs at a concentration of 2.0-2.5×106 cells/mL in 15 mL of complete OptiVitro® NK Cell Expansion Serum-free Medium P01 with OptiVitro® Cytokine III (NK-SFM) supplemented with 10% heat-inactivated autologous plasma.
8. Transfer the cells (from step 7) to the T75 flask (from step 6), add 150 µL of OptiVitro® Cytokine II to the medium, and shake it slightly. Incubate the cells in a humidified 37℃ incubator with an atmosphere of 5% CO2.
9. On Day 3 after NK cell activation, feed the cells with 15 mL of NK-SFM supplemented with 10% heatinactivated autologous plasma.
10. On Day 5 after NK cell activation, feed the cells and adjust the cell concentration to 1.0-1.5×106 cells/mL with NK-SFM supplemented with 5% heat-inactivated autologous plasma.
11. From Day 7 after NK cell activation, feed the cells and adjust the cell concentration to 1.0-1.5×106 cells/mL with NK-SFM supplemented with 5% heat-inactivated autologous plasma every 2-3 days. The cells can be transferred to bioreactors for further expansion at around Day 9-11 after NK cell activation.
