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 106cells/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 106cells/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 106cells/ml之间。
注意：nk扩增阶段，所加新鲜培养基自体血浆含量*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.