Cell culture interfaces for different organ-on-chip applications: from photolithography to rapid-prototyping techniques with sensor embedding

Abstract

Despite the last 60 years have seen major advances in many scientific and technological inputs of drug Research and Development, the number of new molecules hitting the market per billion US dollars of R&D spending has been declined steadily during the same period. The current scenario highlights the need for new research tools to enable reduce costly animal and clinical trials while providing a better prediction about drug efficacy and security in humans A recent emerging approach to improve the current models is emerging from the field of microfluidics, which studies systems that process or manipulate tiny amounts of fluids using channels with dimensions of tens to hundreds of micrometers. Combining microfluidics with cell culture, scientists gave rise to a new field named “Organ-on-chip” (OOC). Microfluidic OOCs are advanced platforms designed to mimic physiological structures and continuous flow conditions, thus allowing the culture of cells in a friendlier microenvironment. This thesis, titled “Cell culture interfaces for different organ-on-chip applications: from photolithography to rapid-prototyping techniques with sensor embedding”, aims to design, simulate and test new OOC devices to reproduce cell culture interface under flow conditions. The work has a focus on the exploration of novel fabrication techniques which enable rapid prototyping of OOC devices, reducing costs, time and human labor associated to the fabrication process. The final objective is to demonstrate the viability of the devices as research tools for biological problems, applying them to the tubular kidney and the blood brain barrier (BBB). To achieve the objective, at least three device version have been developed: 1) OOCv1, fabricated by multilayer PDMS soft lithography; 2) OOCv2, fabricated in thermoplastic by layered object manufacturing using both a vinyl cutter and a laser cutter, integrating standard fluidic connectors alone (OOCv2.1) or together with embedded electrodes (OOCv2.2); 3) OOCv3 using a mixed technique of laser cut and 3D printing by stereolithography. All devices are fabricated using biocompatible materials with high optical quality and an embedded commercial membrane. The biological experiments with renal tubular epithelial cells, realized on OOCv1 and OOCv2.1 devices, demonstrated the viability of the device for culturing cells under flow conditions. The study realized on fatty acid oxidation and accumulation in cells exposed to physiological and diabetogenic oscillating levels of glucose suggest a possible positive role of shear stress in activation of fatty acid metabolism. The studies were performed using a compact experimental unit with embedded flow control which reduce significatively the complexity and cost of the fluidic experimental setup. The biological experiments on the BBB confirmed viability of OOCv2.1 and OOCv2.2 for compartmentalized co-culturing of endothelial cells and pericytes. The formation and recovery of the barrier after disruptive treatment has been assessed using different techniques, including immunostaining, fluorescence and live phase contrast imaging, and electrical impedance spectroscopy. The repeatability of measurements using electrodes was verified. A model to classify measurements from different timepoints has been developed, resulting in accuracy of 100% in learning and 90% in testing case. Results are confirmed by imaging data, which also suggest a critical role of pericytes in the development, maintenance, and regulation of BBB, in accordance with the literature.

En los últimos años está emergiendo una nueva propuesta para mejorar los modelos actuales en el estudio de nuevos fármacos. Mediante la fusión de cultivos celulares y microfluídica ha nacido un nuevo campo de aplicación denominado “Órgano-en-un-chip” (OOC), donde se recrea un entorno fisiológico capaz de reproducir unidades funcionales mínimas de diversos órganos del cuerpo humano. Un elemento importante para el desarrollo de dispositivos OOC es la reproducción de zonas de interacción entre varios tejidos formados por diferentes tipos celulares. Esta tesis, titulada “Interfaces de cultivo celular para diferentes aplicaciones de OOC: desde fotolitografía a técnicas de prototipado rápido con inclusión de sensores”, tiene como objetivo el diseño, simulación y evaluación de dispositivos OOC capaces de reproducir superficies de contacto de tejidos contiguos expuestos a flujo. El trabajo está enfocado a la exploración de nuevas técnicas de fabricación que permitan el prototipado rápido de dispositivos OOC, reduciendo costes, tiempo y mano de obra asociada a dicha fabricación. El objetivo final es demostrar la utilidad de los dispositivos como herramientas de investigación para problemas biológicos, aplicándolos en esta tesis al estudio del túbulo renal y de la barrera hematoencefálica. Para ello se han fabricado tres versiones de dispositivos: 1) OOCv1 fabricado por litografía suave en múltiples capas de PDMS; 2) OOCv2 fabricado con cortadora de vinilo y cortadora láser en múltiples capas de materiales termoplásticos y con electrodos integrados en la versión OOCv2.2; 3) OOCv3 fabricado mediante impresión 3D por esterolitografía. Todos los dispositivos están hechos de materiales biocompatibles de alta calidad óptica, con conectores fluídicos y una membrana comercial integrada. Los experimentos biológicos sobre túbulo renal, realizados en los dispositivos OOCv1 y OOCv2, han demostrado la viabilidad de los dispositivos, integrados con un sistema de flujo, para estudios de la metabolización de ácidos grasos en el riñón relacionados con condiciones diabetogénicas. Los experimentos biológicos sobre la barrera hematoencefálica han confirmado la viabilidad de OOCv2 para el cocultivo compartimentado de células endoteliales de cerebro y pericitos. La integración de electrodos en el OOCv2.2 ha demostrado ser una técnica fiable para la medición de la integridad de barreras biológicas de modo no-invasivo, libre de etiqueta (“label-free”), y a tiempo real gracias a la espectroscopía de impedancia.