Characterization, modification and applicability of an in vitro placental transfer assay for screening purposes

Characterization, modification and applicability of an in vitro placental transfer assay for screening purposes

Caroline Gomes; Catharina W. van Dongen; Barbara Birk; Eric Fabian; Julian Doersam; Bennard van Ravenzwaay; Robert Landsiedel.

Key-words: placental transfer, in vitro models, BeWo b30, embryotoxicity

Embryotoxicity is a toxicological endpoint with high impact in the registration process of chemicals and drugs. Alternative methods to animal testing are being developed for developmental toxicity and are applied already for screening purposes. Nevertheless, transfer of an active substance via the placenta is essential to induce potential direct effects on the embryo. Placental transfer can be investigated in vitro by the determination of the placental transfer rate and the thereof derived apparent permeability coefficient (Papp value) of a substance. Using a trophoblastic cell line (BeWo b30) on a transwell system, a cell barrier is formed separating the apical (representing maternal side) from the basolateral (representing fetal side) compartment. However, varied protocols exist and often insufficient characterization is performed. Therefore, in the present study the main methodology is extensively characterized and optimized to enable screening processes. Characterization parameters are trans-epithelial electrical resistance (TEER), fluorescein transfer (paracellular control), histology, immunohistochemistry of cell tight junctions and transfer of permeability controls amoxicillin (low) and antipyrine (high). The modification alters the frequency of medium change. Stable cell layer integrity was observed on day 6 based on the characterization parameters, which also showed high reproducibility. The TEER increased from day 3 to 6 reaching a value of 46.8 Ω·cm^-2. This increase was followed by a decrease of 80% on the Fluorescein transfer, confirming the barrier integrity. The Papp values of permeability controls amoxicillin and antipyrine were in the literature range for both methodologies. Moreover, several substances, difenoconazole, flusilazole and triadimefon were tested for its placental transfer rate, resulting in Papp values from 7.8 to 41.8 x10⁶ cm/s. These results demonstrate that even compounds from the same chemical class can have different transfer rates. Determining the relative Papp to the reference compound antipyrine facilitates the comparison among substances in different experiments or laboratories. In conclusion, the established protocol may be combined with other in vitro or in silico developmental toxicity assays to support an animal free higher tier testing strategy.

Abstract was published in Naunyn-Schmiedeberg's Arch Pharmacol (2017) 390(Suppl 1)