NK 细胞扩增和活化
1. 抗凝血室温离心，400 xg，10min，上层血浆转移到新管。
2. 热灭活自体血浆，56°C，30min。离心， 800 xg ， 20 min，去除沉淀。4°C 保存上清血浆，培养备用。
3. 加入等体积PBS（不含钙镁）替代被移除血浆保持体积不变，小心重悬血细胞。
注意：PBS预加0.1%人血清白蛋白（HSA）利于保持血细胞活性。
4. 使用淋巴细胞分离液从上面的血样中制备PBMCs（人外周血单个核细胞）。
注意：使用新鲜采集的人血（2h内采集）有利培养，不要使用超过24h的血样。
5. 至少5倍体积的PBS（不含钙镁）清洗PBMCs。室温离心500xg，10min，收集PBMCs。
6. PBMCs稀释到 1 x 106 cells/mL，使用NK活化培养基配比1.8mL NK活化添加剂和10%自体血浆。
7. 预包被T-75培养瓶使用前，用PBS（不含钙镁）清洗两次。
8. 添加30 mL PBMC悬液到预包被T-75培养瓶，37°C，5% CO2培养。
注意：第六天之前，如果培养基变黄，细胞密度超过2.0 x 106 cells/mL，需要添加新鲜的NK活化培养基配比10%自体血浆，保持细胞密度大于 5 x 105 cells/mL。
9. 培养第六天，培养液离心，适量NK扩增培养基（1000 IU/mL IL-2 和10%自体血浆）重悬细胞，再转移到T-225培养瓶或透气细胞培养袋。
10. 每隔两三天，根据细胞扩增状态，培养体系中添加新鲜的NK扩增培养基（1000 IU/mL IL-2 和10%自体血浆）保持细胞密度在5 x 105到 2.0 x 106 cells/mL之间。
注意：NK扩增阶段，所加新鲜培养基自体血浆含量推荐范围：0.5%-1%。
11. 培养14天左右，收获NK细胞。
注意：整个实验过程中，细胞悬液吹打和培养容器拍打都要轻柔，以避免细胞损伤，保持细胞活性。
NK Cell Activation and Expansion
◗◗ Centrifuge the anti-coagulated blood at 400 xg for 10 minutes at room temperature (RT) and then transfer the plasma (on the top layer) into a new tube.
◗◗ Heat inactivate the auto-plasma at 56°C for 30 minutes and then centrifuge at 800 xg for 20 minutes to remove the precipitate. The supernatant plasma should be stored at 4°C, which will be used up in the following 14 culture days.
◗◗ Add equal volume of PBS (Phosphate Buffer Saline without calcium and magnesium, Corning Cat. No. 21-040-CV) as replacement of the removed auto-plasma to maintain a constant volume and resuspend the haemocytes gently.
Note: Pre-adding 0.1% human serum albumin (HSA) in PBS helps to maintain haemocyte viability.
◗◗ Prepare peripheral blood mononuclear cells (PBMCs) from the above blood sample using lymphocyte separation medium (LSM, Corning Cat. No. 25-072-Cl) according to manufacturer’s directions.
Note: Use freshly collected human blood (within 2 hours of collection) for better performance; do not use blood that is drawn more than 24 hours prior to use.
◗◗ Wash the PBMCs with at least 5-fold PBS without calcium and magnesium. Centrifuge at 500 xg for 10 minutes at RT to collect the PBMCs.
◗◗ Dilute the PBMCs to 1 x 106 cells/mL using NK Primary medium containing 1.8 mL NK Primary supplement and 10% auto-plasma.
◗◗ Rinse the pre-coated T-75 flask twice with PBS without calcium and magnesium just before use.
◗◗ Add 30 mL of the PBMC suspension to the rinsed pre-coated T-75 flask and incubate at 37°C in a humidified atmosphere of 5% CO2 in air.
Note: Before Day 6, if the medium turns yellow with the cell density above 2.0 x 106 cells/mL, fresh NK Primary medium plus 10% auto-plasma should be added into the cell suspension to keep the cell density above 5 x 105 cells/mL.
◗◗ On Day 6, centrifuge and resuspend the cells with an appropriate volume of NK Expansion medium containing 1,000 IU/mL IL-2 and 10% auto-plasma and then transfer to a new T-225 flask or gas- permeable culture bag.
◗◗ At an interval of 2 or 3 days, based on the cell proliferation status, add fresh NK Expansion media containing 1,000 IU/mL IL-2 into the culture system and keep the cell density in the recommended density range from 5 x 105 to 2.0 x 106 cells/mL.
Note: In the NK expansion stage, 0.5% to 1% auto-plasma is recommended to be contained in the freshly added media until it has been exhausted.
◗◗ Harvest NK cells around Day 14.
Note: Blow the cell suspension gently and tap the culture vessels softly to avoid cell damage throughout the entire experimental process to maintain cell viability.
