To produce innovative biopharmaceuticals, highly flexible, adaptable, robust, and affordable bioprocess platforms for bioreactors are essential. In this article, we describe the development of a large-area microfluidic bioreactor (LM bioreactor) for mammalian cell culture that works at laminar flow and perfusion conditions. The 184 cm² 32 cisterns LM bioreactor is the largest polydimethylsiloxane (PDMS) microfluidic device fabricated by photopolymer flexographic master mold methodology, reaching a final volume of 2.8 mL. The LM bioreactor was connected to a syringe pump system for culture media perfusion, and the cells’ culture was monitored by photomicrograph imaging. CHO-ahIFN-α2b adherent cell line expressing the anti-hIFN-$a$2b recombinant scFv-Fc monoclonal antibody (mAb) for the treatment of systemic lupus erythematosus were cultured on the LM bioreactor. Cell culture and mAb production in the LM bioreactor could be sustained for 18 days. Moreover, the anti-hIFN-$a$2b produced in the LM bioreactor showed higher affinity and neutralizing antiproliferative activity compared to those mAbs produced in the control condition. We demonstrate for the first-time, a large area microfluidic bioreactor for mammalian cell culture that enables a controlled microenvironment suitable for the development of high-quality biologics with potential for therapeutic use.

La microfluídica y los dispositivos lab-on-a-chip (LOC) permiten la miniaturización y automatización de procesos biológicos complejos a escalas micro y nanométricas. El objetivo de este trabajo de tesis es desarrollar, simular y validar dispositivos microfluídicos aplicados a tres áreas clave: el cultivo de células mamíferas y madre, la producción intensificada de anticuerpos monoclonales (AcMo) y el tamizaje automatizado de fármacos en protozoarios parásitos. Se diseñó e implementó un biorreactor de gran superficie (LM-biorreactor) en PDMS/vidrio para la producción continua de un anticuerpo recombinante anti-hIFN-α2b en células CHO, logrando el doble de constante de afinidad (Ka) en comparación con cultivos en frascos T convencionales. Asimismo, se desarrolló un milli-biorreactor con arquitectura de micropilares para el control pasivo de burbujas en el cultivo de células madre pluripotentes inducidas humanas (hiPSC), manteniendo altos niveles de expresión de los marcadores de pluripotencia Oct4, Nanog y Sox2 durante 5 días. Por último, se aplicó un dispositivo microfluídico dilutor para evaluar 18 compuestos tripanocidas sobre Trypanosoma cruzi mediante seguimiento automatizado de motilidad (software TR-KF), identificando al candidato 2161-X con alta potencia (IC50 = 0,2 µM) e índice de selectividad (SI = 115,6). En conjunto, estos desarrollos ratifican la microfluídica basada en moldes flexográficos de bajo costo y modelado CFD como una plataforma eficiente, escalable y accesible para la biotecnología y el descubrimiento de fármacos.

Chagas disease remains a major neglected tropical disease, highlighting the need for new anti-Trypanosoma cruzi compounds and improved approaches for their phenotypic evaluation. Here, we extended a previously developed Lab-on-a-Chip (LOC) platform integrating a serial dilution generator and automated label-free motility analysis (TR_KF) to the direct screening of extracellular T. cruzi trypomastigotes. The platform was validated in two steps using benznidazole as the reference drug. First, automated motility quantification in conventional 96-well plates was compared with manual Neubauer counting, yielding comparable concentration–response curves and IC₅₀ values. Second, the complete LOC–TR_KF platform was compared with the conventional assay, producing an IC₅₀ that was not significantly different from the reference method. The validated platform was subsequently applied to screen ten candidate samples, identifying combinatorial libraries 2161-X and 2160-X as the most active against trypomastigotes. Comparison with previously reported intracellular amastigote data revealed marked stage-dependent differences in compound activity, while pre-treatment with both libraries reduced parasite infectivity by approximately 50%. Overall, the integrated LOC–TR_KF platform provides pharmacological responses comparable to conventional assays while reducing assay-solution requirements, manual handling and enabling automated, label-free phenotypic screening of T. cruzi trypomastigotes.