Hi everyone,
I am currently culturing primary NK cells, and I use the methods illustrated in the paper : Highly activated and expanded natural killer cells for multiple myeloma immunotherpay in 2012. link as follows:http://www.ncbi.nlm.nih.gov/pubmed/22419581
The method to expand NK cells is : Peripheral blood was centrifuged over Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, NJ, USA). Peripheral blood mononuclear cells were harvested and washed in RPMI
1640 (Invitrogen, Carlsbad, CA, USA) containing 10% fetal
bovine serum (FBS) (Invitrogen). The peripheral blood mononuclear
cells were then co-cultured with irradiated (100 Gray)
K562-mb15-41BBL cells at a ratio of 1.5:1 in RPMI 1640 + 10%
FBS + 300 IU/mL IL2 (Prometheus, San Diego, CA, USA). One
half of the medium was changed every other day with fresh
medium containing IL2. The NK cells were re-stimulated with
K562-mb15-41BBL cells on day 7 and collected on days 10-14 for assays.
What I did was almost the same except that I am changing the whole medium every 2 days. I tested the purity of NK cells on day 7, there's 35% of NK cells among the total CD45+ cells, the paper reported 80% NK cell purity .I was thinking that if there's any problem with what I did. By the way, I sear ched other protocols, they use a specific NK cell expansion medium which was different from normal RPMI1640, if there's anything different, is there any one have the same experience? Thank you
In general, NK cells thrive better when crowded and medium slightly yellowish, so I wouldn't change the whole medium. In addition, IMDM is better than RPMI 1640. Try also to supplement with 2% Humans AB serum in addition to 10% FCS. Make sure all cells are free from mycoplasma.
I agree with Kalle.
Nevertheless, the amount of expanded NK cells in these type of cultures may vary from one individual to another.
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioscience