Understanding poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel as a multifunctional membrane in microfluidic cell culture platform

Cell culture technology developed at the turn of 20th century using Petri dish, which is not able to consider the microenvironment that the cells experience in vessels, has remained virtually unchanged for almost a century. However, such microenvironment associated with cell culture which usually consists of soluble factors, extracellular matrix cues, and cellular networks is difficult to reproduce experimentally with the traditional approach. In order to further elaborate complex mechanisms of cell biology through mimicking such microenvironment in vivo, the technical approaches together with developed microdevices are highly demanded within such a context. Microfluidic devices have been extensively developed and used for cell culture in the last two decades, which offer numerous advantages and a great potential for accurate and efficient control of the complex culturing microenvironment at cellular length scale. However, these devices are relatively complex in their fabrication and integration to fulfil multifunctional tasks for cell culture and drug testing simultaneously, which for example requires a membrane between the culture chamber and drug delivery reservoir to control microenvironment at cellular scale. This thesis is to primarily focus on the feasibility and reliability in the attempt of using poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel as an inserted membrane, based on its permeable and flexible tissue-like properties. PHEMA membrane is able to serve dual purposes in the microfluidic systems in cell culture: i) exchanging nutrients between culture chamber and drug delivery reservoir; and ii) sealing the microchannel circuits.</div

Flow-induced order-order transitions in amyloid fibril liquid crystalline tactoids

Understanding and controlling the director field configuration, shape, and orientation in nematic and cholesteric liquid crystals is of fundamental importance in several branches of science. Liquid crystalline droplets, also known as tactoids, which spontaneously form by nucleation and growth within the biphasic region of the phase diagram where isotropic and nematic phases coexist, challenge our current understanding of liquid crystals under confinement, due to the influence of anisotropic surface boundaries at vanishingly small interfacial tension and are mostly studied under quiescent, quasi-equilibrium conditions. Here, we show that different classes of amyloid fibril nematic and cholesteric tactoids undergo out-of-equilibrium order-order transitions by flow-induced deformations of their shape. The tactoids align under extensional flow and undergo extreme deformation into highly elongated oblate shapes, allowing the cholesteric pitch to decrease as an inverse power law of the tactoids aspect ratio. Energy functional theory and experimental measurements are combined to rationalize the critical elongation ratio above which the director-field configuration of tactoids transforms from bipolar and uniaxial cholesteric to homogenous and to debate on the thermodynamic nature of these transitions. Our findings suggest new opportunities in designing self-assembled liquid crystalline materials where structural and dynamical properties may be tuned by non-equilibrium phase transitions

Integrating Biophysics in Toxicology

Integration of biophysical stimulation in test systems is established in diverse branches of biomedical sciences including toxicology. This is largely motivated by the need to create novel experimental setups capable of reproducing more closely in vivo physiological conditions. Indeed, we face the need to increase predictive power and experimental output, albeit reducing the use of animals in toxicity testing. In vivo, mechanical stimulation is essential for cellular homeostasis. In vitro, diverse strategies can be used to model this crucial component. The compliance of the extracellular matrix can be tuned by modifying the stiffness or through the deformation of substrates hosting the cells via static or dynamic strain. Moreover, cells can be cultivated under shear stress deriving from the movement of the extracellular fluids. In turn, introduction of physical cues in the cell culture environment modulates differentiation, functional properties, and metabolic competence, thus influencing cellular capability to cope with toxic insults. This review summarizes the state of the art of integration of biophysical stimuli in model systems for toxicity testing, discusses future challenges, and provides perspectives for the further advancement of in vitro cytotoxicity studies

Non-linear modeling of active biohybrid materials

Recent advances in engineered muscle tissue attached to a synthetic substrate motivates the development of appropriate constitutive and numerical models. Applications of active materials can be expanded by using robust, non-mammalian muscle cells, such as those of Manduca sexta. In this study, we propose a model to assist in the analysis of biohybrid constructs by generalizing a recently proposed constitutive law for Manduca muscle tissue. The continuum model accounts (i) for the stimulation of muscle fibers by introducing multiple stress-free reference configurations for the active and passive states and (ii) for the hysteretic response by specifying a pseudo-elastic energy function. A simple example representing uniaxial loading-unloading is used to validate and verify the characteristics of the model. Then, based on experimental data of muscular thin films, a more complex case shows the qualitative potential of Manduca muscle tissue in active biohybrid constructs

Blood particulate analogue fluids: A review

Microfluidics has proven to be an extraordinary working platform to mimic and study blood flow phenomena and the dynamics of components of the human microcirculatory system. However, the use of real blood increases the complexity to perform these kinds of in vitro blood experiments due to diverse problems such as coagulation, sample storage, and handling problems. For this reason, interest in the development of fluids with rheological properties similar to those of real blood has grown over the last years. The inclusion of microparticles in blood analogue fluids is essential to reproduce multiphase effects taking place in a microcirculatory system, such as the cell-free layer (CFL) and Fähraeus–Lindqvist effect. In this review, we summarize the progress made in the last twenty years. Size, shape, mechanical properties, and even biological functionalities of microparticles produced/used to mimic red blood cells (RBCs) are critically exposed and analyzed. The methods developed to fabricate these RBC templates are also shown. The dynamic flow/rheology of blood particulate analogue fluids proposed in the literature (with different particle concentrations, in most of the cases, relatively low) is shown and discussed in-depth. Although there have been many advances, the development of a reliable blood particulate analogue fluid, with around 45% by volume of microparticles, continues to be a big challengeThis research was funded by the Spanish Ministry of Science and Education Grant No. PID2019-108278RB-C32 / AEI / 10.13039/501100011033, and Junta de Extremadura (Spain) Grant Nos. GR18175 and IB18005 (partially financed by FEDER funds). The authors also acknowledge the Fundação para a Ciência e a Tecnologia (FCT) for partially financing the research under the strategic grants UIDB/04077/2020, UIDB/00532/2020, and the project NORTE-01-0145-FEDER030171 (PTDC/EME-SIS/30171/2017) funded by COMPETE2020, NORTE 2020, PORTUGAL 2020, Lisb@2020, and FEDE

Microfluidic converging/diverging channels optimised for homogeneous extensional deformation

In this work we optimise microfluidic converging/diverging geometries in order to produce constant strain-rates along the centreline of the flow, for performing studies under homogeneous extension. The design is examined for both two-dimensional and three-dimensional flows where the effects of aspect ratio and dimensionless contraction length are investigated. Initially, pressure driven flows of Newtonian fluids under creeping flow conditions are considered, which is a reasonable approximation in microfluidics, and the limits of the applicability of the design in terms of Reynolds numbers are investigated. The optimised geometry is then used for studying the flow of viscoelastic fluids and the practical limitations in terms of Weissenberg number are reported. Furthermore, the optimisation strategy is also applied for electro-osmotic driven flows, where the development of a plug-like velocity profile allows for a wider region of homogeneous extensional deformation in the flow field

Development of experimental setups for the characterization of the mechanoelectrical coupling of cells in vitro

The field of mechanobiology emerged from the many evidences that mechanical forces acting on cells have a central role in their development and physiology. Cells, in fact, convert such forces into biochemical activities and gene expression in a process referred as mechanotransduction. In vitro models that mimic cell environment also from the mechanical point of view represent therefore a key tool for modelling cell behaviour and would find many applications, e.g. in drug development and tissue engineering. In this work I introduce novel tools for the study of mechanotransduction. In particular, I present a system for the evaluation of the complex response of electrically active cells, such as neurons and cardiomyocytes. This system integrates atomic force microscopy, extracellular electrophysiological recording, and optical microscopy in order to investigate cell activity in response to mechanical stimuli. I also present cell scaffolds for the in vitro study of cancer. Obtained results, although preliminary, show the potential of the proposed systems and methods to develop accurate in vitro models for mechanobiology studies

The Role of Cellular Architecture in Vascular Smooth Muscle Function and Mechanics

University of Minnesota Ph.D. dissertation. August 2017. Major: Biomedical Engineering. Advisor: Patrick Alford. 1 computer file (PDF); x, 98 pages.Recently, there has been a push towards clinical translation of biomechanical models of tissues by developing patient-specific models to predict disease outcomes. To accomplish this, it is necessary to understand the functional and mechanical properties of all the tissue components, including individual cells. In vasculature, tissues and cells have different structures based on their functional role. The principle goal of this work is to determine how cellular architecture influences function and mechanical properties. To test our hypotheses, we have developed in vitro models to study the relationship between structure and function at the tissue and cellular scale. We have developed microfluidic capture array device (MCAD) technology to study cell structure and function in 2D engineered vascular smooth muscle tissue and have developed cellular micro-biaxial stretching (CμBS) microscopy to determine single cell mechanical properties. First, using MCAD technology we were able to vary initial cell-cell contact during seeding to bias the cellular architecture in confluent vascular smooth muscle tissues. We found that tissues seeded using initially higher cell–cell contact conditions yielded tissues with more elongated cellular architecture which lead to greater contractile function in engineered tissues. We then used CμBS microscopy to determine the elastic anisotropic mechanical properties of individual cells, given by the strain energy density (SED) function. We found that smooth muscle cells (VSMCs) with native-like architectures are highly anisotropic and can be described by a SED based on the actin cytoskeletal organization. Then, we utilized CμBS microscopy to characterize loading and unloading mechanics of VSMCs. We found that VSMCs exhibit architecture-dependent anisotropic hysteresis where highly structured VSMCs exhibit typical hysteresis associated with viscous loss when stretched in the direction of actin fiber alignment but exhibit reverse hysteresis when stretched in the direction orthogonal to actin fiber alignment. We then modeled the observed hysteresis using two models: a quasi-linear (QLV) model and a Hill-type active fiber model and found that the QLV model was insufficient to characterize the anisotropic hysteresis but the Hill-type active fiber model was able to predict the anisotropic hysteresis in highly-organized VSMCs