HELP! CTL lines won't expand!
jladasky at pmgm.Stanford.EDU
Sat Jun 20 17:40:11 EST 1998
I'm trying to establish alloreactive T cell lines, using the
cell line Daudi transfected with beta-2 microglobulin (Daudi is b2m-neg-
ative). I've been attempting to use a protocol that was developed in
our lab for use with Class I-transfected 721.221 B-lymphoblastoid cell
lines as stimulators (721.221 is Class I-negative). The person who has
developed the protocol has left the lab. I am in touch with her by
email, but I can't send her microscope pictures of my cultures...
I expand cells in bulk for two weeks, with stimulation at days
zero and seven. At day 14 I sort the CD3+/CD8+ cells, and seed them
at various dilutions (typically 50 cells/well, 10 cells/well) on Terasaki
plates. Each well on the Terasaki plate contains 10,000 irradiated self
PBMC, 2,000 irradiated Daudi/b2m stimulators, and 20 U/ml IL-2 (in 20
microliters). Wells are boosted at day 21 with 20 additional microliters
containing cells and IL-2 as above. By about day 25, some of the wells
are showing growth. I replace 20 microliters of medium with fresh IL-2
medium at this time. By day 28, I start expanding wells -- first to 100
microliters in a single well on a U-bottom 96-well plate, then to two
wells, and then to a 48-well flat-bottom plate. I increase volumes,
self-PBMC feeders, and stimulators accordingly.
Here's where I run into trouble. Although I've had about 30
wells on the Terasaki plates showing growth, every time I transfer the
cells most of the cultures die *fast* (within 48 hours). So I got about
15 of the cultures to the 96-well plate, four of these to two wells, and
only one to a 48-well plate. Now it looks like that one hardy culture
is also dying. Typically, the cell medium has turned a *little* orange
when I decide to expand the culture, but it doesn't look bad.
Yesterday I looked at *nine* protocols for CTL line expansion.
It seems like some of the primary culture steps in our protocol (days
0-14) are a bit unusual (and I think I understand the reasons for these),
but nothing in the secondary steps seems to be that unusual. And the
secondary cultures are where I'm getting my problems anyway. _Current_
_Protocols_in_Molecular_Biology_ suggests that one should alternate be-
tween providing stimulators with the weekly PBMC and withholding the stim-
ulators. They also suggest regular Ficoll purification of the live cells
away from the dead cells. None of the papers I read went to such extreme
measures -- and besides, is it practical to do 30+ Ficolls at once?
Am I over-stimulating my cells, leading to apoptosis? Am I under-
stimulating them? What's up?
I will post or email more details of the protocol if it will
help you to understand my problem. I need cells, and soon! I've started
a new batch of cells, and will be sorting a week from today.
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw. - John Ladasky
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