Sofia Apodaca ¹ Marco D. Salvatore ¹ Arturo Muñoz-Calderón ¹ María De Los Ángeles Curto ¹ Silvia A. Longhi ¹ Alejandro G. Schijman ^1,2*

• ¹ CONICET Instituto de Investigaciones en Ingeniería Genética y Biología Molecular Dr. Héctor N. Torres (INGEBI), Buenos Aires, Buenos Aires, Argentina
• ² National Scientific and Technical Research Council (CONICET), Buenos Aires, Argentina

Human trophoblastic cell lines, like BeWo, are commonly employed in 2D models for placental Trypanosoma cruzi infection studies. While T. cruzi induces trophoblast differentiation in these models, they inadequately represent natural infections. Three dimensional (3D) microtissue cultures have emerged as physiologically relevant in-vitro models, mimicking tissue microarchitecture and providing an environment closer to natural infections. These 3D cultures exhibit functions such as cell proliferation, differentiation, morphogenesis, and gene expression resembling in vivo situations.In this study, we developed a 3D culture model using the BeWo cell line and nonadherent agarose molds created with the MicroTissues® 3D Petri Dish® system. Both Small and Large models (12-256 and 12-81) were tested, varying initial cell numbers. Seeding 1000 BeWo cells per microwell in the Large model resulted in comparable cellular viability to 2D cultures, with a theoretical diameter of 408.68 ± 12.65 µm observed at 5 days. Functionality, assessed through βhCG production, exceeded levels in 2D cultures at both 3 and 5 days. T. cruzi infection was demonstrated through microscopy and qPCR, using three parasite strains (VD, K98, and Pan4) with distinct discrete typing units and validated virulence levels. This innovative 3D model provides a simple yet effective approach for generating viable and functional cultures susceptible to T. cruzi infection, presenting significant potential for studying the placental microenvironment.