The importance of metrological tools to implementation of alternative method OECD TG 428.

Usually, if percutaneous absorption tests are conducted in accordance with OECD Guideline 428, in vitro determination is accepted by mainly regulatory agencies. In this paper, we focus on the lack of comparability of the results regarding the permeation parameter/flow rate, although it is widely discussed in the literature. This work sought to evaluate the absorption of caffeine using Franz-type diffusion cell with porcine ear skin samples, varying the storage duration and the way to handle them. Metrological tools were used for caffeine quantification such as certified reference material candidate, calibrated instruments, and validated methodology. Our results corroborate with the recommendation that membranes should be freshly prepared or frozen for short periods. Samples frozen for approximately one year should not be used because they present high cutaneous absorption. The results obtained for the absorption rate (J) are comparable to the results obtained by previous studies using similar experimental conditions. The evidence of the barrier characteristic promoted by the stratum corneum and the effect promoted by the storage time is shown through J = 6.25 ± 0.48 µg/cm2/h. We demonstrated the importance of metrological tools to guarantee reproducibility and comparability of the results between different laboratories.

Dissecting coronary angiogenesis: 3D co-culture of cardiomyocytes with endothelial or mesenchymal cells.

In embryogenesis, coronary blood vessels are formed by vasculogenesis from epicardium-derived progenitors. Subsequently, growing or regenerating myocardium increases its vasculature by angiogenesis, forming new vessels from the pre-existing ones. Recently, cell therapies for myocardium ischemia that used different protocols have given promising results, using either extra-cardiac blood vessel cell progenitors or stimulating the cardiac angiogenesis. We have questioned whether cardiomyocytes could sustain both vasculogenesis and angiogenesis. We used a 3D culture model of tissue-like spheroids in co-cultures of cardiomyocytes supplemented either with endothelial cells or with bone marrow-derived mesenchymal stroma cells. Murine foetal cardiomyocytes introduced into non-adherent U-wells formed 3D contractile structures. They were coupled by gap junctions. Cardiomyocytes segregated inside the 3D structure into clumps separated by connective tissue septa, rich in fibronectin. Three vascular endothelial growth factor isoforms were produced (VEGF 120, 164 and 188). When co-cultured with human umbilical cord endothelial cells, vascular structures were produced in fibronectin-rich external layer and in radial septa, followed by angiogenic sprouting into the cardiomyocyte microtissue. Presence of vascular structures led to the maintenance of long-term survival and contractile capacity of cardiac microtissues. Conversely, bone marrow mesenchymal cells formed isolated cell aggregates, which progressively expressed the endothelial markers von Willebrand's antigen and CD31. They proceeded to typical vasculogenesis forming new blood vessels organised in radial pattern. Our results indicate that the in vitro 3D model of cardiomyocyte spheroids provides the two basic elements for formation of new blood vessels: fibronectin and VEGF. Within the myocardial environment, endothelial and mesenchymal cells can proceed to formation of new blood vessels either through angiogenesis or vasculogenesis, respectively.