Corning® NK细胞活化扩增培养套装操作方法

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.