Corning® NK細(xì)胞活化擴(kuò)增培養(yǎng)套裝操作方法
NK 細(xì)胞擴(kuò)增和活化
1. 抗凝血室溫離心，400 xg，10min，上層血漿轉(zhuǎn)移到新管。
2. 熱滅活自體血漿，56°C，30min。離心， 800 xg ， 20 min，去除沉淀。4°C 保存上清血漿，培養(yǎng)備用。
3. 加入等體積PBS（不含鈣鎂）替代被移除血漿保持體積不變，小心重懸血細(xì)胞。
注意：PBS預(yù)加0.1%人血清白蛋白（HSA）利于保持血細(xì)胞活性。
4. 使用淋巴細(xì)胞分離液從上面的血樣中制備PBMCs（人外周血單個核細(xì)胞）。
注意：使用新鮮采集的人血（2h內(nèi)采集）有利培養(yǎng)，不要使用超過24h的血樣。
5. 至少5倍體積的PBS（不含鈣鎂）清洗PBMCs。室溫離心500xg，10min，收集PBMCs。
6. PBMCs稀釋到 1 x 106 cells/mL，使用NK活化培養(yǎng)基配比1.8mL NK活化添加劑和10%自體血漿。
7. 預(yù)包被T-75培養(yǎng)瓶使用前，用PBS（不含鈣鎂）清洗兩次。
8. 添加30 mL PBMC懸液到預(yù)包被T-75培養(yǎng)瓶，37°C，5% CO2培養(yǎng)。
注意：第六天之前，如果培養(yǎng)基變黃，細(xì)胞密度超過2.0 x 106 cells/mL，需要添加新鮮的NK活化培養(yǎng)基配比10%自體血漿，保持細(xì)胞密度大于 5 x 105 cells/mL。
9. 培養(yǎng)第六天，培養(yǎng)液離心，適量NK擴(kuò)增培養(yǎng)基（1000 IU/mL IL-2 和10%自體血漿）重懸細(xì)胞，再轉(zhuǎn)移到T-225培養(yǎng)瓶或透氣細(xì)胞培養(yǎng)袋。
10. 每隔兩三天，根據(jù)細(xì)胞擴(kuò)增狀態(tài)，培養(yǎng)體系中添加新鮮的NK擴(kuò)增培養(yǎng)基（1000 IU/mL IL-2 和10%自體血漿）保持細(xì)胞密度在5 x 105到 2.0 x 106 cells/mL之間。
注意：NK擴(kuò)增階段，所加新鮮培養(yǎng)基自體血漿含量推薦范圍：0.5%-1%。
11. 培養(yǎng)14天左右，收獲NK細(xì)胞。
注意：整個實(shí)驗(yàn)過程中，細(xì)胞懸液吹打和培養(yǎng)容器拍打都要輕柔，以避免細(xì)胞損傷，保持細(xì)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.
