HELP! CTL lines won't expand!

John Ladasky jladasky at pmgm.Stanford.EDU
Sat Jun 20 17:40:11 EST 1998

Hi folks,

	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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