Primary Porcine Brain Endothelial Cells as In Vitro Model to Study Effects of Ultrasound and Microbubbles on Blood-Brain Barrier Function.

Focused ultrasound (FUS) in the presence of microbubbles transiently and reversibly opens the blood-brain barrier (BBB) in rodents and humans, thereby providing a time window for increased drug delivery into brain tissue. To get insight into the underlying mechanisms that govern ultrasound (US)-mediated opening of the BBB, in vitro models are a useful alternative. In this paper, we have utilized an in vitro BBB model that consists of primary porcine brain endothelial cells (PBECs). PBEC monolayers are grown on permeable membranes, which allow assessment of key features of BBB function as well as US treatment. This experimental model is characterized by low permeability for both small molecules and proteins, has a high transendothelial electrical resistance, and expresses tight junctions and efflux pumps. Here, we compare the effects of inertial and stable cavitation in the presence of SonoVue microbubbles on PBEC monolayers' electrical resistance and permeability properties. Our results point out the fragility of PBEC monolayers, which enhances results variability. In particular, we show that handling of the inserts, such as medium change and transfer to the US setup, modifies the cellular response, and immunostaining of the monolayers introduces damage and cell detachment within the US-exposed monolayers. Our results indicate that stable cavitation might have a more pronounced impact on cell permeability as compared with inertial cavitation in vitro. This paper might contribute to further development of experimental setups that are suitable to characterize the impact of FUS and microbubbles on BBB properties in vitro.

Uptake, penetration, and binding of monoclonal antibodies with increasing affinity in human osteosarcoma multicell spheroids.

BACKGROUND: Multicell spheroids from the human osteosarcoma cell line, OHS, were incubated with increasing concentrations of the monoclonal antibodies TP-1, TP-3 and 9.2.27 having different affinities (Ka = 8.5 x 10(8) M-1, 3.4 x 10(9) M-1 and 1.4 x 10(11) M-1, respectively). MATERIALS AND METHODS: Uptake and penetration of the fluorescein labelled antibodies were studied using confocal laser scanning fluorescence microscopy, and antibodies bound per cell were measured using flow cytometry. RESULTS: The antibody with highest affinity, 9.2.27, bound to all available binding sites in one cell-layer before reaching the next, whereas TP-3 and TP-1 gradually bound to an increasing number of epitopes of all cells throughout the spheroids. The penetration rate and antibody uptake increased with increasing antibody concentration up to saturating concentrations. 9.2.27 required saturation concentrations to reach the center of the spheroids, whereas TP-1 and TP-3 penetrated the centre when concentrations below saturation were applied. CONCLUSION: The antibodies might be useful in radioimmunotherapy of micrometastases.

Penetration and binding of monoclonal antibody in human osteosarcoma multicell spheroids. Comparison of confocal laser scanning microscopy and autoradiography.

Penetration and binding of monoclonal antibody (MAb) in multicell osteosarcoma spheroids have been studied by autoradiography and confocal laser scanning microscopy (CLSM). Optical sectioning of the 3-dimensional spheroids was performed by CLSM. Owing to attenuation of fluorescence intensity, FITC-labelled MAb could not be detected at depths greater than 60 microm within the spheroids. The antibody uptake seen in autoradiographs and CLSM images 60 microm within the spheroids were essentially identical. MAb had reached all parts of the spheroids within 6 h. Quantitative measurements of the fluorescence intensity of FITC-labelled MAb seen in confocal images and measurements of MAb bound per cell using flow cytometry, showed that maximum uptake was reached after 6 h. The possibility to perform both quantitative and qualitative measurements makes CLSM a promising method for studying antibody uptake in thick tissue samples.