Hello John, I have dealt extensively with the generation of human T cell
lines and clones. First, let me state, all though the books say it should
be possible, maintaning long term cultures of the clones is very
difficult. The best bet is to get them to high numbers, freeze, and work
from these stocks with as few passages as possible.
I can anticipate what went through your head. I can not get them to high
numbers. I personally have found that this is difficult and the cells
often crash after a couple of stimulations and rest. We have found that
the addition of 4U/ml of rhIL-7 helps in this regard. Look up the
literature on it. Cells are not supposed to lose specificity with IL-7.
10-20 u/ml of IL-2 is the best range to work with, higher doses does keep
them alive longer and expands them greater, but then you have the problem
of loosing specificity. In mice 100 U/ml of IL-2 starts turning them into
non-specific LAK cells.
After the first stimulation and rest cycle, keep the IL-2 in the cells
during the rest and not in the stimulation for the next 2 cycles, after
that keep IL-2 in always. Add the Il-7, if not at the start, add it to
the first rest and keep it from then on.
Ficol about once every 2-3 weeks. Keeps the debris down. You might also
want to add fresh media every couple of days.
Remember, you may want to try thinking of ways to test what you need to
test fast, after the 2nd stimulation. Human T cells are not happy without
all the support factors (adhesion and cytokine).
I hope I was a help
TLWPHD
