Pitingolo, Gabriele (2016) Engineered microfluidic platforms for microenvironment control and cell culture. [Tesi di dottorato]

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Tipologia del documento:	Tesi di dottorato
Lingua:	English
Titolo:	Engineered microfluidic platforms for microenvironment control and cell culture
Autori:	Autore Email Pitingolo, Gabriele gabriele.pitingolo@unina.it
Data:	31 Marzo 2016
Numero di pagine:	184
Istituzione:	Università degli Studi di Napoli Federico II
Dipartimento:	Ingegneria Chimica, dei Materiali e della Produzione Industriale
Scuola di dottorato:	Ingegneria industriale
Dottorato:	Ingegneria dei materiali e delle strutture
Ciclo di dottorato:	28
Coordinatore del Corso di dottorato:	nome email Mensitieri, Giuseppe giuseppe.mensitieri@unina.it
Tutor:	nome email Netti, Paolo Antonio [non definito] Vecchione, Raffaele [non definito]
Data:	31 Marzo 2016
Numero di pagine:	184
Parole chiave:	Microfluidics,Tissue on a chip, Blood brain barrier, Nanoparticles, Microfabrication
Settori scientifico-disciplinari del MIUR:	Area 09 - Ingegneria industriale e dell'informazione > ING-IND/34 - Bioingegneria industriale
Depositato il:	13 Apr 2016 00:13
Ultima modifica:	04 Mag 2017 01:00
URI:	http://www.fedoa.unina.it/id/eprint/11029

Abstract

The aim of this thesis is to overcome present limitations in mimicking the in vivo cellular microenvironments with novel in vitro cell culture systems. Since microtechnologies and microfluidics, in particular, provide the tools to reproduce in vivo-like cellular microenvironments in vitro, current relevant research is presented and areas where more research is needed in characterizing the in vitro microenvironment are outlined. 2D and 3D dynamic cell-culture models have recently garnered great attention because they often promote levels of cell differentiation and tissue organization not possible in conventional 2D static culture systems. In this work we developed new microfabrication approaches to reproduce cell-culture microenvironments that both support tissue differentiation and recapitulate the tissue–tissue interfaces, spatiotemporal chemical gradients, and mechanical microenvironments of living systems. These ‘tissues-on-chips’ permit the study of human physiology in physiological contest, enable development of novel in vitro disease models, and could potentially serve as replacement for animals used in drug development and toxin testing. To this aim, a blood brain barrier (BBB) microfluidic device was designed based on a transparent polyester porous membrane sandwiched between a top and a bottom overlying channel made of PMMA. According to our results, the PMMA is the most suitable biocompatible material for the porous membrane integration between two layers, compared to other materials, such as PDMS, commonly used to fabricate similar devices. We faced its permeability issue by engineering the proposed device with a collecting chamber, in the top part, to ensure the oxygen provision. In order to verify the efficacy of this microfluidic system, we tested the passage of BSA and nanoparticles compared to blank porous filter. Afterwards, in order to better mimic the blood-brain barrier and its circular shape we developed an innovative method to fabricate miniaturized circular microchannels from square geometry. A wide range of perfusable microvessel models have been developed, exploiting advances in microfabrication, microfluidics, biomaterials, stem cell technology, and tissue engineering. These models vary in complexity and physiological relevance, but provide a diverse tool kit for the study of vascular phenomena and methods to vascularize artificial organs. Here we developed a fast, cheap and reproducible method to fabricate circular microchannels by coupling spin coating with micromilled square microchannels. In order to validate our approach, an endothelial cell layer was formed by culturing brain endothelial bEnd.3 cells inside the proposed circular microchannels. In addition, considering that the diameter of blood vessels, in humans, spans more than four orders of magnitude, from about 8 μm in capillaries to more than 1 cm in large elastic arteries, we developed a low cost approach, using gelatin dehydration as intermediate, to fabricate microchannels of 5-8 microns in width, in order to mimic smaller capillaries. We are currently exploring the possibility to apply our approaches to fabricate a 3D microvessel model, totally in gelatin, to better mimic the extracellular matrix and the endothelium. In parallel, associated with the development of the above described devices, we proposed practical tips to the miniaturization community, developing innovative techniques that offer a solution to commonly encountered problems in the microfabrication field, or improvements (e.g. a simplification) on existing techniques. Thanks to their cost and technical advantages these microfluidic platforms may have extensive applications for neurobiology, cancer biology and for studying the cell-biomaterial interaction.

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