Biopreservation and Biobanking - Ziath - 18

18
ANGEL ET AL.
FIG. 5. Reduction of antigen-specific T cell response. (A) Shows reduction of T cell response after stimulation with CEF
peptide pool and (B) after stimulation with CMV peptide pool. To show the reduction in a clearer way, a linear fit was
performed and is presented as a line. Figures show reduction of immune response after 50-350 temperature cycles in
comparison with 0 cycles. Figures show mean - standard deviation of four donors. The gradients are significantly different
from zero temperature cycles with p< 0.05.
We observed a decrease of recovery and viability after
overnight culture in comparison with the measurement directly
after thawing. This may not only be due to normal
apoptosis and necrosis processes during overnight culture,25,29,45,46
but it may also be caused by preapoptotic
cells, which are triggered by cryopreservation to induce
apoptosis during overnight culture.23 This difference did not
change with increasing numbers of cycles (data not shown).
Moreover, Smith et al. showed by simulating transport
conditions that the number of apoptotic cells increases and
additionally the T cell response decreases.26
The T cell response is a crucial factor in vaccine development.29
Therefore, reliable performance of the ELISpot
assay is important. Thus, we also investigated the influence
of suboptimal storage on T cell functionality. We observed
a high T cell response for the control cells stored below
-130C without temperature fluctuations. Furthermore, we
observed that the T cell response of the cells, which were
exposed to the temperature rises, decreased with increasing
numbers of temperature cycles. Due to the expected variability
when working with cell suspensions together with the
small intervals of 50 temperature cycles, not every interval
data group significantly differs from the one before or after.
Nevertheless, the linear regression analysis, statistically
and graphically, shows a significant reduction of the
antigen-specific T cell response over increasing number of
temperature cycles. We suspect that changes of cell surface
molecules and changes of expression levels, which were
described by Costantini et al. (2003), are also triggered by
the temperature rises from below -130Cupto -60C.47
Furthermore, Kreher et al. showed that cytokine secretion
changed due to freezing and thawing processes.38 This could
explain the observed reduction in T cell response in ELISpot.
Above all, the maintenance of T cell functionality during
cryopreservation is controversially discussed. Regarding the
T cell immune response, there are different observations.
Meanwhile, Owen et al. noticed the loss of T cell response,
and Kreher et al. observed the maintenance of antigenspecific
T cell response.25,38
We suspect that the storage conditions may potentially be
responsible for the different observations. Thus, for optimal
storage conditions, it is important to avoid temperature fluctuations.
The detection and documentation of deviations in
deep temperature storage play a crucial role, especially in
terms of storage duration and sample history. Samples, which
are often stored for several years, could potentially degrade
with regard to various examined parameters. Moreover,
sample history regarding temperature fluctuations in longterm
storage is most often unknown. To understand this retrospectively,
the temperature should be recorded over longer
periods in regular short intervals. It would be an improvement
toward standardization and optimal cryopreservation if updated
quality management systems and standard operating
procedures would be established.
Although the cell suspension did not thaw completely, but
merely warmed up to -60C, the temperature rises sufficed
to lead to multiple cell damages. In summary, we showed
that temperature rises during deep temperature storage lead
to reduction of viability, recovery, and T cell functionality
of PBMCs. The study emphasizes the need for controlled
storage, especially in clinical studies and biobanks, to yield
comparable and reliable results and to maintain the quality
of cryopreserved samples.
Furthermore, to guarantee controlled storage and handling
without interruption of the cooling chain, protective hood
systems, wherein the working area for sample removal and
sorting is cooled down, are recommended. This would reduce
the temperature fluctuations to a minimum. Moreover,
establishment of an updated quality management system and
introduction of standard operating procedures wherein,
among others, deviations are documented would lead to a
better standardization and quality of the respective samples.
In the future, more attention should be paid to the storage
conditions to achieve a high recovery, maintain the viability,
and the T cell functionality of cells. Moreover, studies are
needed to determine whether other cell types such as stem
cells and other biological materials react in a similar way to
temperature cycles during cryostorage. In addition, further

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