Parametric investigation of static and dynamic cell culture conditions and their impact on hCMEC/D3 barrier properties.

In brain research, the hCMEC/D3 cell line is widely used for the establishment of a human in vitro blood-brain barrier (BBB) model. However, its barrier integrity seems to be insufficient for drug permeability studies, represented by rather low transendothelial electrical resistance (TEER) and high permeability of small molecules. Therefore, this study covers a parametric investigation of static and dynamic cell culture conditions to improve barrier functionality of hCMEC/D3. The effect of basal media was investigated by analyzing changes in proliferation rate, barrier integrity and gene expression of cellular junction proteins. The cells were able to grow in different cell culture media, including serum-free media. However, none of these media enhanced strongly the growth rate or barrier integrity compared to the microvascular endothelial cell growth medium-2 (EGM™-2 MV). Furthermore, hCMEC/D3 cells did not respond positively regarding TEER to any tested parameter neither supplements, coating materials nor co-cultures with the human immortalized astrocyte cell line SVGmm. Furthermore, the impact of dynamic conditions was examined by using the Dynamic Micro Tissue Engineering System (DynaMiTES). Cultivation conditions were successfully adapted to the DynaMiTES design and no negative effect was detected by analyzing cell viability and cell count, albeit TEER remained also unchanged. Consequently, the hCMEC/D3 model has considerable limitations and further improvements or alternative cell lines are required.