3 research outputs found
Design, characterization and validation of integrated bioelectronics for cellular studies: from inkjet-printed sensors to organic actuators
Mención Internacional en el título de doctorAdvances in bioinspired and biomimetic electronics have enabled
coupling engineering devices to biological systems with unprecedented
integration levels. Major efforts, however, have been devoted to interface
malleable electronic devices externally to the organs and tissues. A promising
alternative is embedding electronics into living tissues/organs or,
turning the concept inside out, lading electronic devices with soft living
matters which may accomplish remote monitoring and control of tissue’s
functions from within. This endeavor may unleash the ability to engineer
“living electronics” for regenerative medicine and biomedical applications.
In this context, it remains a challenge to insert electronic devices efficiently
with living cells in a way that there are minimal adverse reactions
in the biological host while the electronics maintaining the engineered
functionalities. In addition, investigating in real-time and with minimal
invasion the long-term responses of biological systems that are brought
in contact with such bioelectronic devices is desirable.
In this work we introduce the development (design, fabrication and
characterization) and validation of sensors and actuators mechanically
soft and compliant to cells able to properly operate embedded into a
cell culture environment, specifically of a cell line of human epithelial
keratinocytes. For the development of the sensors we propose moving from conventional microtechnology approaches to techniques compatible
with bioprinting in a way to support the eventual fabrication of tissues
and electronic sensors in a single hybrid plataform simultaneously. For
the actuators we explore the use of electroactive, organic, printing-compatible
polymers to induce cellular responses as a drug-free alternative
to the classic chemical route in a way to gain eventual control of biological
behaviors electronically. In particular, the presented work introduces
inkjet-printed interdigitated electrodes to monitor label-freely and
non-invasively cellular migration, proliferation and cell-sensor adhesions
of epidermal cells (HaCaT cells) using impedance spectroscopy and the
effects of (dynamic) mechanical stimulation on proliferation, migration
and morphology of keratinocytes by varying the magnitude, frequency
and duration of mechanical stimuli exploiting the developed biocompatible
actuator.
The results of this thesis contribute to the envision of three-dimensional
laboratory-growth tissues with built-in electronics, paving exciting
avenues towards the idea of living smart cyborg-skin substitutes.En los útimos años los avances en el desarrollo de dispositivos
electrónicos diseñados imitando las propiedades de sistemas vivos han
logrado acoplar sistemas electrónicos y órganos/tejidos biológicos con
un nivel de integración sin precedentes. Convencionalmente, la forma
en que estos sistemas bioelectrónicos son integrados con órganos o tejidos
ha sido a través del contacto superficial entre ambos sistemas, es
decir acoplando la electrónica externamente al tejido. Lamentablemente
estas aproximaciones no contemplan escenarios donde ha habido una
pérdida o daño del tejido con el cual interactuar, como es el caso de daños
en la piel debido a quemaduras, úlceras u otras lesiones genéticas
o producidas. Una alternativa prometedora para ingeniería de tejidos y
medicina regenerativa, y en particular para implantes de piel, es embeber
la electrónica dentro del tejido, o presentado de otra manera, cargar
el sistema electrónico con células vivas y tejidos fabricados por ingeniería
de tejidos como parte innata del propio dispositivo. Este concepto
permitiría no solo una monitorización remota y un control basado en
señalizaciones eléctricas (sin químicos) de tejidos biológicos fabricados
mediante técnicas de bioingeniería desde dentro del propio tejido, sino
también la fabricación de una “electrónica viva”, biológica y eléctricamente
funcional. En este contexto, es un desafío insertar de manera
eficiente dispositivos electrónicos con células vivas sin desencadenar
reacciones adversas en el sistema biológico receptor ni en el sistema
electrónico diseñado. Además, es deseable monitorizar en tiempo real
y de manera mínimamente invasiva las respuestas de dichos sistemas
biológicos que se han añadido a tales dispositivos bioelectrónicos.
En este trabajo presentamos el desarrollo (diseño, fabricación y caracterización)
y validación de sensores y actuadores mecánicamente suaves y
compatibles con células capaces de funcionar correctamente dentro de un
entorno de cultivo celular, específicamente de una línea celular de células epiteliales
humanas. Para el desarrollo de los sensores hemos propuesto utilizar
técnicas compatibles con la bioimpresión, alejándonos de la micro fabricación
tradicionalmente usada para la manufactura de sensores electrónicos, con el
objetivo a largo plazo de promover la fabricación de los tejidos y los sensores
electrónicos simultáneamente en un mismo sistema de impresión híbrido.
Para el desarrollo de los actuadores hemos explorado el uso de polímeros
electroactivos y compatibles con impresión y hemos investigado el efecto
de estímulos mecánicos dinámicos en respuestas celulares con el objetivo a
largo plazo de autoinducir comportamientos biológicos controlados de forma
electrónica. En concreto, este trabajo presenta sensores basados en electrodos
interdigitados impresos por inyección de tinta para monitorear la migración
celular, proliferación y adhesiones célula-sustrato de una línea celular de
células epiteliales humanas (HaCaT) en tiempo real y de manera no invasiva
mediante espectroscopía de impedancia. Por otro lado, este trabajo presenta
actuadores biocompatibles basados en el polímero piezoeléctrico fluoruro de
poli vinilideno y ha investigado los efectos de estimular mecánicamente células
epiteliales en relación con la proliferación, migración y morfología celular
mediante variaciones dinámicas de la magnitud, frecuencia y duración de
estímulos mecánicos explotando el actuador biocompatible propuesto.
Ambos sistemas presentados como resultado de esta tesis doctoral
contribuyen al desarrollo de tejidos 3D con electrónica incorporada,
promoviendo una investigación hacia la fabricación de sustitutos equivalentes
de piel mitad orgánica mitad electrónica como tejidos funcionales
biónicos inteligentes.The main works presented in this thesis have been
conducted in the facilities of the Universidad Carlos III
de Madrid with support from the program Formación del
Profesorado Universitario FPU015/06208 granted by Spanish Ministry
of Education, Culture and Sports. Some of the work has been also
developed in the facilities of the Fraunhofer-Institut für Zuverlässigkeit
und Mikrointegration (IZM) and University of Applied Sciences (HTW) in
Berlin, under the supervision of Prof. Dr. Ing. H-D. Ngo during a research
visit funded by the Mobility Fellows Program by the Spanish Ministry of
Education, Culture, and Sports.
This work has been developed in the framework of the projects
BIOPIELTEC-CM (P2018/BAA-4480), funded by Comunidad de Madrid,
and PARAQUA (TEC2017-86271-R) funded by Ministerio de Ciencia e
Innovación.Programa de Doctorado en Ingeniería Eléctrica, Electrónica y Automática por la Universidad Carlos III de MadridPresidente: José Antonio García Souto.- Secretario: Carlos Elvira Pujalte.- Vocal: María Dimak
Real-Time Impedance Monitoring of Epithelial Cultures with Inkjet-Printed Interdigitated-Electrode Sensors
From electronic devices to large-area electronics, from individual cells to skin substitutes, printing techniques are providing compelling applications in wide-ranging fields. Research has thus fueled the vision of a hybrid, printing platform to fabricate sensors/electronics and living engineered tissues simultaneously. Following this interest, we have fabricated interdigitated-electrode sensors (IDEs) by inkjet printing to monitor epithelial cell cultures. We have fabricated IDEs using flexible substrates with silver nanoparticles as a conductive element and SU-8 as the passivation layer. Our sensors are cytocompatible, have a topography that simulates microgrooves of 300 µm width and ~4 µm depth, and can be reused for cellular studies without detrimental in the electrical performance. To test the inkjet-printed sensors and demonstrate their potential use for monitoring laboratory-growth skin tissues, we have developed a real-time system and monitored label-free proliferation, migration, and detachment of keratinocytes by impedance spectroscopy. We have found that variations in the impedance correlate linearly to cell densities initially seeded and that the main component influencing the total impedance is the isolated effect of the cell membranes. Results obtained show that impedance can track cellular migration over the surface of the sensors, exhibiting a linear relationship with the standard method of image processing. Our results provide a useful approach for non-destructive in-situ monitoring of processes related to both in vitro epidermal models and wound healing with low-cost ink-jetted sensors. This type of flexible sensor as well as the impedance method are promising for the envisioned hybrid technology of 3D-bioprinted smart skin substitutes with built-in electronics.The work by D.M.-M. has been performed in the frame of an FPU Program, FPU015/06208, and a Mobility Fellows Program, both granted by the Spanish Ministry of Education, Culture and Sports. This work has been funded by the Comunidad de Madrid under the grant BIOPIELTEC-CM (P2018/BAA-4480) and the Ministerio de Ciencia e Innovación under the grant PARAQUA (TEC2017-86271-R)
Design, implementation, and validation of a piezoelectric device to study the effects of dynamic mechanical stimulation on cell proliferation, migration and morphology
Cell functions and behavior are regulated not only by soluble (biochemical) signals but also by biophysical and mechanical cues within the cells’ microenvironment. Thanks to the dynamical and complex cell machinery, cells are genuine and effective mechanotransducers translating mechanical stimuli into biochemical signals, which eventually alter multiple aspects of their own homeostasis. Given the dominant and classic biochemical-based views to explain biological processes, it could be challenging to elucidate the key role that mechanical parameters such as vibration, frequency, and force play in biology. Gaining a better understanding of how mechanical stimuli (and their mechanical parameters associated) affect biological outcomes relies partially on the availability of experimental tools that may allow researchers to alter mechanically the cell’s microenvironment and observe cell responses. Here, we introduce a new device to study in vitro responses of cells to dynamic mechanical stimulation using a piezoelectric membrane. Using this device, we can flexibly change the parameters of the dynamic mechanical stimulation (frequency, amplitude, and duration of the stimuli), which increases the possibility to study the cell behavior under different mechanical excitations. We report on the design and implementation of such device and the characterization of its dynamic mechanical properties. By using this device, we have performed a preliminary study on the effect of dynamic mechanical stimulation in a cell monolayer of an epidermal cell line (HaCaT) studying the effects of 1 Hz and 80 Hz excitation frequencies (in the dynamic stimuli) on HaCaT cell migration, proliferation, and morphology. Our preliminary results indicate that the response of HaCaT is dependent on the frequency of stimulation. The device is economic, easily replicated in other laboratories and can support research for a better understanding of mechanisms mediating cellular mechanotransduction.Peer ReviewedPostprint (published version