12.3.3.4 Culture Medium and the Use

12.3.3.4 Culture Medium and the Use of Fresh Versus Cryopreserved PBMC
The culture medium seems to be important and could be responsible for
differences in activation capacity. Laan et al. (1998) showed that proliferation was
optimal when using RPMI as culture medium, whereas PBMC cultured in Yssel
medium showed better cytokine responses. Yssel medium was first used as a
serum-free medium based on IMDM that induced proliferative responses in mixed
lymphocyte cultures that are comparable to those obtained in medium containing
serum (Yssel et al., 1984). IMDM contains many energy sources and in
combination with the glucose in the human AB serum and the sodium pyruvate in
IMDM, the cultured cells are provided with a much larger energy source than
RPMI-1640 medium alone.
Another important aspect when dealing with in vitro stimulation of PBMC is
the use of fresh cells compared to cryopreserved cells. Cryopreservation is in
some cases unavoidable when working in the field or when handling large batches
of patient samples. An advantage of cryopreservation is that thawing and culturing
can be performed on a convenient moment and more samples can be thawed at
once. This minimizes the operator-dependent interassay variability. However,
cryopreservation could cause a small delay in the activation of the cryopreserved
PBMC and could reduce the cytokine levels when stimulating with certain stimuli
(Jeurink et al., 2008). Monocytes can be successfully cryopreserved with different
protocols, with recoveries ranging from 50% to 75% and afterwards stimulated to cytokine production (e.g., IL-10) which appeared not significantly altered
compared to fresh PBMC (Best et al., 2007). It is therefore advisable to choose the
same methods for all analyses as this enables better comparison of the data
obtained for individual PBMC donors, and allergens.
An advantage of the PBMC-model is that the results can be ascribed to innate
and adaptive immune responses, based on cell specific responses. The response of
the cells can be measured over time. After 1