Abstract Book 2010 - CIMT Annual Meeting

019 Buhl | Therapeutic vaccination
Cryopreservation of high-concentrated peripheral blood mononuclear
cells by controlled-rate freezer results in higher cell
yields for dendritic cell-based immunotherapy
Timo Buhl 1 , Anke Schardt 1 , Tobias Legler 2 , Michael P. Schön 1 , Holger A. Hänßle 1
1 Department of Dermatology and
2 Department of Transfusion Medicine of Georg August University, Göttingen, Germany
Cryopreservation of immature or mature dendritic
cells (DC) has been described as a suitable method
to achieve large numbers of DC for immunotherapeutic
trials against cancer. Recently it was shown
that cryopreservation of peripheral blood mononuclear
cells (PBMC) with subsequent differentiation
into DC is superior compared to cryopreservation
of immature or mature DC in terms of resulting DC
quantity and immuno-stimulating capacity. The
aim of our study was to establish an optimized
protocol for the cryopreservation of highly-concentrated
PBMC for DC-based immunotherapy. Cryopreserved
cell preparations were analyzed regarding
recovery, viability, phenotype, and functional
properties. Results were compared to fresh DC
generated instantly from the same donor. In contrast
to PBMC frozen by standard isopropyl alcohol
freezing containers, PBMC cryopreservation in an
automated controlled-rate freezer (CRF) resulted
in significantly higher cell yields of immature and
mature DC after thawing and subsequent differentiation
to DC. The CRF is connected to liquid nitrogen,
allowing the freezing program to exactly
control and adjust the temperature of the freezing
chamber. Therefore, the crystallization heat at the
freezing point of the sample can be compensated
by a sharp temperature decrease in the freezing
chamber. Immature DC yields and total protein
content after using CRF were comparable to freshly
generated DC and exceeded results of standard isopropyl
alcohol freezing by approximately 50%. DC
generated after both freezing protocols of PBMC revealed
no relevant phenotypic or functional differences.
Our analyses included phenotypic markers,
allogenic T cell stimulatory assays, viability tests
and microarray cytokine profiles. The latter showed
that few cytokines were differently secreted during
DC maturation depending on the method of cryopreservation.
The majority of cytokine levels was
not altered in correlation with the method of cryopreservation.
In summary, automated controlled
rate freezing of PBMC represents a much improved
freezing method capable of increasing DC yields for
cancer immunotherapy.
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