Microtechnologies for Cell Microenvironment Control and Monitoring

A great breadth of questions remains in cellular biology. Some questions cannot be answered using traditional analytical techniques and so demand the development of new tools for research. In the near future, the development of highly integrated microfluidic analytical platforms will enable the acquisition of unknown biological data. These microfluidic systems must allow cell culture under controlled microenvironment and high throughput analysis. For this purpose, the integration of a variable number of newly developed micro- and nano-technologies, which enable control of topography and surface chemistry, soluble factors, mechanical forces and cell-cell contacts, as well as technology for monitoring cell phenotype and genotype with high spatial and temporal resolution will be necessary. These multifunctional devices must be accompanied by appropriate data analysis and management of the expected large datasets generated. The knowledge gained with these platforms has the potential to improve predictive models of the behavior of cells, impacting directly in better therapies for disease treatment. In this review, we give an overview of the microtechnology toolbox available for the design of high throughput microfluidic platforms for cell analysis. We discuss current microtechnologies for cell microenvironment control, different methodologies to create large arrays of cellular systems and finally techniques for monitoring cells in microfluidic devices.E.A.-H. acknowledges funding from the Basque Government, Department of Education, for predoctoral fellowship 2016. M.G.-H. acknowledges funding from the University of the Basque Country UPV/EHU, PIF16/204 predoctoral fellowship "call for recruitment of research personnel in training". J.E.-E. acknowledges funding from the University of the Basque Country UPV/EHU, postdoctoral fellowship ESPPOC 16/65 "Call for recruitment and specialization of Doctor Researchers 2016". M.M.D.P. and L.B.-D., acknowledge funding support from University of the Basque Country UPV/EHU, UFI11/32, and from Gobierno Vasco under Grupos Consolidados with Grant No. IT998-16. F.B.-L. acknowledges funding support from the Ramon y Cajal Programme (Ministerio de Economia y Competitividad), Spain. F.B.-L. and L.B.-D. acknowledge funding support from the European Union's Seventh Framework Programme (FP7) for Research, Technological Development and Demonstration under Grant agreement No. 604241 as well as Gobierno Vasco, Dpto. Industria, Innovacion, Comercio y Turismo under ELKARTEK 2015 with Grant No. KK-2015/0000088

Electrospun micro/nanodevices for controlled biomolecule release

Controlled biomolecule release technology represents one of the fastest advancing areas of science and engineering. For instance, in drug delivery area, such release system offers numerous advantages compared to conventional dosage drug forms including improved efficiency, reduced toxicity and controlled release profile. Current challenges in this area include biocompatiblity and biodegrability of the materials used in the system, controllablity and effectivity of the control mechanism, easiness of device fabraiction and drug loading loss as well as total cost. In this work, a simple and effective method is adopted to design and fabricate controlled release devices employing smart conmtrol mechanism. Such a technology could be further applied in pharmaceutics, biomeidical science and biotechnologies. Controlled molecule release devices in this work employ the advantage of core-shell structures. In the first design, core-shell microcapsules are developed capable of regulating the release profile of encapsulated molecules. These microcapsules uniquely contain embedded miniature actuators inside their liquid core. The internal actuators are made of stimuli-responsive smart hydrogel beads. The embedded hydrogel beads swell in response to external electric fields, regulating the internal pressure of the liquid core, and thus the diffusion rate, of the encapsulated molecules from the microcapsules. The incorporation of the actuators into the interior of the microcapsules provides an internal control variable to a conventional diffusion-based release process. The microcapsules, which behave much like micro-electro-mechanical systems (MEMS), are fabricated by a simple co-electrospray process. This fabrication technique allows integrating the hydrogel beads, forming the polymer shell, and loading the releasable molecules simultaneously in one step. The other controlled release device is developed by embedding nanofluidic biomolecule reservoirs into a polymer network of a stimuli-responsive hydrogel. The reservoirs are made of liquid core-polymer shell nanofibers using co-electrospinning technique. The mechanism of controlled release is based on buckling instability of the polymer shell under combined axial and radial compression, caused by volume changes of hydrogel responding to a specific external stimulus. The device decouples releasable biomolecules from a hydrogel polymer matrix, avoiding chemical interactions between biomolecules and hydrogel polymer chains, and thus, alleviating nontrivial chemical and biological engineering design of hydrogel formulations. Temperature-sensitive hydrogel is used as a model hydrogel

Multiphoton techniques for dynamic manipulation of cellular microenvironments

textA multitude of biophysical signals, including chemical, mechanical, and contact guidance cues, are embedded within the extracellular matrix (ECM) to dictate cell behavior and determine cell fate. To understand the complexity of the cell-matrix interaction and how changes to the ECM contribute to the development of tissues or diseases, three-dimensional (3D), culture systems that can decouple the effects of these cues on cell behavior are required. This dissertation describes the development and characterization of approaches based on multiphoton excitation (MPE) to control the chemical, mechanical, and topographical presentation of micro-3D-printed (μ-3DP) protein hydrogels independently. Protein hydrogels were chemically functionalized via the MPE-induced conjugation of benzophenone-biotin without altering the physical properties of the matrix. Complex, immobilized patterns and chemical gradients were generated within protein hydrogels with a high degree of spatial resolution in all axes. Hydrogel surfaces were also labeled with adhesive moieties to promote localized Schwann cell adhesion and polarization. Laser shrinking, a method based on MPE to manipulate the topographical and mechanical presentation of protein hydrogels after fabrication, is also presented. Topographical features on an originally flat substrate are created with depths approaching 6 μm. The Young’s modulus of protein hydrogels can also be increased by 6-fold (~15 – ~90 kPa) using laser shrinking, and parameters can be adjusted to create continuous gradient profiles for studying durotaxis. At determined scan conditions, the two properties can be adjusted independently of each other. Most importantly, the physical properties of the hydrogels can be manipulated in situ to study the effects of dynamic changes to the substrates on cells. As a potential tool to monitor cellular responses to presented cues, fluorescent probes that detect nitric oxide are characterized. Collectively, these technologies represent a key advance in hydrogel tunability, as the platforms presented offer independent, dynamic, and spatiotemporal control of the chemical, mechanical, and topographical features of protein hydrogels. The introduced technologies expand the possibilities of protein hydrogels to clarify underlying factors of cell-matrix interactions that drive morphogenesis and pathogenesis, and are broadly applicable to a multitude of physiological systems.Chemical Engineerin

Reactive inkjet printing and functional inks : a versatile route to new programmed materials

Starting as an ink dispensing tool for documents and images, inkjet printing has emerged as an important instrument for delivering reactive fluids, into a means for creating new, programmed materials. Inkjet is a processing technology with some very unique capabilities, which allows the handling of materials in the picoliter range, and the creation of functionality in new, previously unexplored ways. In particular, drop-on-demand technology provides the chance to dispense liquids in picoliter/nanoliter quantities to very specific locations, with minimal material loss, and in a contact-free manner. This dramatic scale-down of production, not just miniaturization but "nanonization", affords materials that would be either too costly or otherwise inaccessible by other manners. As this is still an emerging technology, there remain a lot of opportunities to pioneer new applications. The underlying, unifying concept behind the chapters of this thesis has been an interest in investigating how inkjet printing, combined with reactive inks, can lead to new applications, new devices, and new materials, wherein unique functionality is imparted as a direct result of the confluence between microfluidic processing, chemistry, and life science. The ability to deliver uniform, sub-nanoliter droplets to specific locations opens up new possibilities that did not exist before. Inherent in the geometry of such droplets, the volume of liquid dispensed also offers some special utility. Based on the aforementioned diameters, drop-on-demand inkjet printing can deliver volumes in the range of approximately 0.5 to 1,000 pL; the direct writing attributes of inkjet ensure that these droplets are not only precise, but can be delivered to a specific location, giving them a "home address". This combination of precise, reproducible, small aliquots and precision deposition is especially important for preparing high-density analytical arrays, as discussed in Chapter 1 on inkjet printing of proteins. In the case of highly specialized proteins such as reactive enzymes or antibodies, where available materials are often limited, the ability to dispense precise quantities in a reproducible fashion means that small amounts of precious material can be used parsimoniously to perform thousands of experiments without compromising the quality of the data. For drop-on-demand printing, droplets normally produced by inkjet printing are commonly in the range of 10 to 125 µm in diameter, depending on the physical characteristics of the fluid, the nozzle used, and the printing conditions; taking advantage of the this aliquot size has some unique attributes that make dispensing highly suitable to materials science challenges that have gone unmet. In the second chapter of this thesis, this size domain is taken advantage of for use in tissue engineering, where it is used to create soft, cell-scale porogenic structures by the use of a reversible, rapid alginate gelation reaction to freeze droplet structures in place. By switching to a continuous inkjet device, larger volumes of beads in the size domain of 100 to 500 µm can be achieved, opening up prospects for pore sizes matching those needed for hosting capillaries. By incorporating reversible hydrogels as a motif in these applications, these controlled cell-scale dimensions can be retained during key processing steps, and then removed (or eroded) later after they have served their function. Extending the concept to the task of dispensing living cells, in Chapter 3, printed alginate structures are used for cell encapsulation. By adjusting the printing conditions to prevent jet break-up before alginate hardening, continuous, one-dimensional "living threads" can be created, which allow for cell cultures to be handled and woven into desired complex patterns. In addition to their role as basic building materials for tissue engineering scaffolds, the alginate threads provide a stable, bio-friendly environment for culturing different cell types, with cells exhibiting a high post-processing viability rate. In Chapter 4, the lower limits of single cell printing are explored, in the concept of "one cell-one well", where the attributes of inkjet printing are used to dispense individual cells. By careful selection of droplet size and accounting of cell concentrations, the statistical probability of single cell printing can be optimized, yielding spatially addressable arrays of isolated living cell cultures on a surface. Additional steps necessary to prevent cells from dehydration are also outlined, offering access to high density arrays of isolated living single cells on glass slides, where each individual droplet acts as independent nanoincubator, hosting intrinsically monoseptic cell cultures in parallel. In addition to describing the theoretical limits of single-pass cell printing experiment designs, an outline is given for experimental designs for tuning single particulate dispensing probability to any value desired between 0 and 1. The focus of Chapter 5 relates to reactive inkjet printing of ultrathin films on surfaces. For systems with moderately good surface wetting, such as polar solvents on glass or metal oxides, inkjet printed droplets result in features ranging approximately from 20 to 300 µm in diameter per droplet. By first printing a thiol-functionalized heterochelic linker and covalently bonding it to the print surface, the surface will accommodate subsequent thiol-ene click reactions only with original monolayer, and only where the first and second deposition features overlap. This combination of spatial selectivity as well as chemoselectivity allows for the preparation of a wide range of monolayers on a printed surface, in a format well-suited to automated surface characterization techniques, as was illustrated using XPS. In Chapters 6, two different categories of irreversible polymerization reactions are described, where print features are reacted in a specific pattern that is process unique. Printable ionogels are developed, which impart conductivity to printed patterns, and consequently, functionality to only those locations where the material has been deposited. Also in Chapter 6, the first example of a moisture-sensitive reactive printing is outlined, where a diisocyanate is combined with different polyols within seconds to create highly crosslinked, ultra-stiff surfaces, which can be built up into three dimensions by successive layering. The topics outlined in this thesis are intended to illustrate the breadth of how inkjet technology can be utilized to support a diverse field of materials science applications — particularly when coupled with modular, off-the-shelf synthetic transformations. The incorporation of synthetic chemistry into inkjet extends the application of inkjet from dispensing static materials merely from a cartridge onto a target, into a dynamic tool for transforming these materials into something new. At the same time, inkjet printing and other allied microfluidics tools enable chemistry experiments (and by extension, life science experiments) on a scale that would otherwise be challenging to realize by other means. The two driving forces of high-throughput experiment design, miniaturization and automation, are both embodied in this dispensing technique, and consequently inkjet printing is a rapidly evolving discipline; it is the intent of this work and the examples given to underscore the diversity offered by this technology

Engineering patterned and dynamic surfaces for the spatio-temporal control of cell behaviour

Stem cell shape and mechanical properties in vitro can be directed by geometrically defined micropatterned adhesion substrates. However conventional methods are limited by the fixed micropattern design, which cannot recapitulate the dynamic changes of the natural cell microenvironment. Recent advancements in microfabrication technologies in combination with the use of light-responsive materials, allow to manipulate the shape of living cells in real-time in a non-invasive Spatio-temporal controlled way to introduce additional geometrically defined adhesion sites and to study relative cell behaviour. Here, the confocal laser technique is exploited for dynamically evaluate the variation over time of the tensional and morphological cell state. This method allows the precise control of specific actin structures that regulate cell architecture. Actin filament bundles, initially randomly organized in circular-shaped cells, are induced to align and distribute to form a rectangular-shaped cell in response to specific dynamic changes in the cell adhesion pattern. The changes in morphology also reflect dramatic changes in FAs distribution, cell mechanics, nuclear morphology, and chromatin conformation. The reported strategy is convenient to explore the cell-substrate interface and the mechanisms through which cell geometry regulates cell signalling in a facile and cost-effective manner and it open new routes to understand how the field of dynamic platforms should potentially contribute to unveil complex biological events such as the modulation of cell shape

Nanoparticles in polyelectrolyte multilayer layer-by-layer (LbL) films and capsules : key enabling components of hybrid coatings

Originally regarded as auxiliary additives, nanoparticles have become important constituents of polyelectrolyte multilayers. They represent the key components to enhance mechanical properties, enable activation by laser light or ultrasound, construct anisotropic and multicompartment structures, and facilitate the development of novel sensors and movable particles. Here, we discuss an increasingly important role of inorganic nanoparticles in the layer-by-layer assembly—effectively leading to the construction of the so-called hybrid coatings. The principles of assembly are discussed together with the properties of nanoparticles and layer-by-layer polymeric assembly essential in building hybrid coatings. Applications and emerging trends in development of such novel materials are also identified

Complex 3D microfluidic architectures formed by mechanically guided compressive buckling.

Microfluidic technologies have wide-ranging applications in chemical analysis systems, drug delivery platforms, and artificial vascular networks. This latter area is particularly relevant to 3D cell cultures, engineered tissues, and artificial organs, where volumetric capabilities in fluid distribution are essential. Existing schemes for fabricating 3D microfluidic structures are constrained in realizing desired layout designs, producing physiologically relevant microvascular structures, and/or integrating active electronic/optoelectronic/microelectromechanical components for sensing and actuation. This paper presents a guided assembly approach that bypasses these limitations to yield complex 3D microvascular structures from 2D precursors that exploit the full sophistication of 2D fabrication methods. The capabilities extend to feature sizes <5 μm, in extended arrays and with various embedded sensors and actuators, across wide ranges of overall dimensions, in a parallel, high-throughput process. Examples include 3D microvascular networks with sophisticated layouts, deterministically designed and constructed to expand the geometries and operating features of artificial vascular networks

Photoresponsive azobenzene-based materials for smart cell culture applications

The work described in this thesis intends to exploit the photomechanical properties of azobenzene-based materials for the realization of functional photoresponsive cell culture platforms. Up to date, a big variety of photolithographic techniques has been successfully adopted for azopolymer photopatterning and even the possibility of a dynamic modulation of the topographical features of 2D cell culture substrates appeared to be feasible using these materials. In fact, a great biological interest resides in the dynamic modulation of the cell-material crosstalk, in order to recapitulate in vitro the fast remodeling of the natural extracellular microenvironment, in terms of biochemical, topographical and mechanical cues. In this thesis, light-induced topographical and structural modifications of different azobenzene-based materials have been used in many biological applications, either at a single-cell level, or in multicellular systems. In particular, light-based techniques already used for single-cell investigations have been implemented to study more complex biological processes, which involve the cell-cell interactions in their final biological response. At the same time, the realization of novel 3D photoresponsive platforms for smart cell culture has been introduced. Findings discussed in this thesis add a valuable contribution to the field of azobenzene-based matters as novel dynamic cell-instructive materials