Transfection of rat brain endothelium in a primary culture model of the blood-brain barrier at different states of barrier maturity

Central nervous system diseases are becoming more prevalent. Unfortunately, the treatment of CNS diseases is often rendered complicated by the inability of many drugs of therapeutic relevance to cross the blood-brain barrier (BBB). In order to enhance drug delivery to the brain, different approaches have been developed. Gene therapy could be a promising and novel approach to overcome the restricting properties of the BBB to polypeptides and proteins. Gene therapy is based on the delivery of genetic material into brain capillary endothelial cells (BCECs), which, theoretically, will result in expression and secretion of the recombinant protein from the BCECs and into the brain, thus turning BCECs into small recombinant protein factories.
In this study, the possibility of using BCECs as small factories for recombinant protein production was investigated. To mimic the in-vivo situation as closely as possible, an in-vitro BBB model was established by isolating primary BCECs, pericytes, and astrocytes from rats. The model was subsequently characterized by immunostaining, RT-qPCR, and measurements of trans-endothelial electrical resistance (TEER). Previous studies have stated that efficient gene transfection is positively correlated with cell division, with dividing cells being more sensitive to gene transfection. Therefore, by using this model, the transfection efficiency of the rat endothelium was investigated in an immature state and mature state of the BBB. During the transfection period, measurements of TEER were performed to study variations in BBB tightness. The transfection efficiency was investigated using RT-qPCR and immunostaining.