Cell-based Devices Research Articles

Microfluidic In Vitro Platform for (Nano)Safety and (Nano)Drug Efficiency Screening.

Microfluidic technology is a valuable tool for realizing more in vitro models capturing cellular and organ level responses for rapid and animal-free risk assessment of new chemicals and drugs. Microfluidic cell-based devices allow high-throughput screening and flexible automation while lowering costs and reagent consumption due to their miniaturization. There is a growing need for faster and animal-free approaches for drug development and safety assessment of chemicals (Registration, Evaluation, Authorisation and Restriction of Chemical Substances, REACH). The work presented describes a microfluidic platform for in vivo-like in vitro cell cultivation. It is equipped with a wafer-based silicon chip including integrated electrodes and a microcavity. A proof-of-concept using different relevant cell models shows its suitability for label-free assessment of cytotoxic effects. A miniaturized microscope within each module monitors cell morphology and proliferation. Electrodes integrated in the microfluidic channels allow the noninvasive monitoring of barrier integrity followed by a label-free assessment of cytotoxic effects. Each microfluidic cell cultivation module can be operated individually or be interconnected in a flexible way. The interconnection of the different modules aims at simulation of the whole-body exposure and response and can contribute to the replacement of animal testing in risk assessment studies in compliance with the 3Rs to replace, reduce, and refine animal experiments.

CRISPR-addressable yeast strains with applications in human G protein–coupled receptor profiling and synthetic biology

Genome stability is essential for engineering cell-based devices and reporter systems. With the advent of CRISPR technology, it is now possible to build such systems by installing the necessary genetic parts directly into an organism's genome. Here, we used this approach to build a set of 10 versatile yeast-based reporter strains for studying human G protein-coupled receptors (GPCRs), the largest class of membrane receptors in humans. These reporter strains contain the necessary genetically encoded parts for studying human GPCR signaling in yeast, as well as four CRISPR-addressable expression cassettes, i.e. landing pads, installed at known safe-harbor sites in the yeast genome. We showcase the utility of these strains in two applications. First, we demonstrate that increasing GPCR expression by incrementally increasing GPCR gene copy number potentiates Gα coupling of the pharmacologically dark receptor GPR68. Second, we used two CRISPR-addressable landing pads for autocrine activation of a GPCR (the somatostatin receptor SSTR5) with its peptide agonist SRIF-14. The utility of these reporter strains can be extended far beyond these select examples to include applications such as nanobody development, mutational analysis, drug discovery, and studies of GPCR chaperoning. Additionally, we present a BY4741 yeast strain created for broad applications in the yeast and synthetic biology communities that contains only the four CRISPR-addressable landing pads. The general utility of these yeast strains provides an inexpensive, scalable, and easy means of installing and expressing genes directly from the yeast genome to build genome-barcoded sensors, reporter systems, and cell-based factories.

Copper Metal Organic Polyhedron (Cu-MOP) Hydrogel as Responsive Cytoprotective Shell for Living Cell Encapsulation.

Single-cells coated with functional shells to protect them from external harsh condition have great potential applications in many fields such as tissue engineering, cell-based devices, cell biology, and so on. Herein, copper metal organic polyhedron (Cu-MOP) hydrogel has been applied as a soft shell for cell protection under both physical and chemical stimulations. Compared with a previous strategy, this MOP-Gel shell not only possesses more satisfied protection effect but also could be prepared and removed facilely without any damage to the encapsulated cells.

Elucidation and refinement of synthetic receptor mechanisms.

Synthetic receptors are powerful tools for engineering mammalian cell-based devices. These biosensors enable cell-based therapies to perform complex tasks such as regulating therapeutic gene expression in response to sensing physiological cues. Although multiple synthetic receptor systems now exist, many aspects of receptor performance are poorly understood. In general, it would be useful to understand how receptor design choices influence performance characteristics. In this study, we examined the modular extracellular sensor architecture (MESA) and systematically evaluated previously unexamined design choices, yielding substantially improved receptors. A key finding that might extend to other receptor systems is that the choice of transmembrane domain (TMD) is important for generating high-performing receptors. To provide mechanistic insights, we adopted and employed a Förster resonance energy transfer-based assay to elucidate how TMDs affect receptor complex formation and connected these observations to functional performance. To build further insight into these phenomena, we developed a library of new MESA receptors that sense an expanded set of ligands. Based upon these explorations, we conclude that TMDs affect signaling primarily by modulating intracellular domain geometry. Finally, to guide the design of future receptors, we propose general principles for linking design choices to biophysical mechanisms and performance characteristics.

Modelling Cell Origami via a Tensegrity Model of the Cytoskeleton in Adherent Cells.

Cell origami has been widely used in the field of three-dimensional (3D) cell-populated microstructures due to their multiple advantages, including high biocompatibility, the lack of special requirements for substrate materials, and the lack of damage to cells. A 3D finite element method (FEM) model of an adherent cell based on the tensegrity structure is constructed to describe cell origami by using the principle of the origami folding technique and cell traction forces. Adherent cell models contain a cytoskeleton (CSK), which is primarily composed of microtubules (MTs), microfilaments (MFs), intermediate filaments (IFs), and a nucleoskeleton (NSK), which is mainly made up of the nuclear lamina and chromatin. The microplate is assumed to be an isotropic linear-elastic solid material with a flexible joint that is connected to the cell tensegrity structure model by spring elements representing focal adhesion complexes (FACs). To investigate the effects of the degree of complexity of the tensegrity structure and NSK on the folding angle of the microplate, four models are established in the study. The results demonstrate that the inclusion of the NSK can increase the folding angle of the microplate, indicating that the cell is closer to its physiological environment, while increased complexity can reduce the folding angle of the microplate since the folding angle is depended on the cell types. The proposed adherent cell FEM models are validated by comparisons with reported results. These findings can provide theoretical guidance for the application of biotechnology and the analysis of 3D structures of cells and have profound implications for the self-assembly of cell-based microscale medical devices.

Steamed cake-derived 3D carbon foam with surface anchored carbon nanoparticles as freestanding anodes for high-performance microbial fuel cells

Anode design is highly significant for microbial fuel cells, since it simultaneously serves as the scaffold for electroactive microorganisms and as a medium for electron migration. In this study, a stiff 3D carbon foam with surface anchored nitrogen-containing carbon nanoparticles was facilely constructed via in-situ polyaniline coating of carbonized steamed cake prior to the carbonization process. The resultant product was determined to be an excellent freestanding anode that enabled the microbial fuel cell to deliver a maximum power density of up to 1307 mW/m2, which significantly outperformed its non-coated counterpart, the widely used commercial carbon felt. Further investigations revealed that the overall performance enhancement was associated with the open porosity, enlarged electroactive surface, increased biocompatibility, and decreased electric resistance of the anode scaffold. This promising anode material would offer a green and economical option for fabricating high-performance microbial fuel cell-based devices towards various ends.

HMSCs from UCB: Isolation, Characterization and Determination of Osmotic Properties for Optimal Cryopreservation

In tissue engineering, storing of biological material represents a fundamental step to bring cell-based medical devices to market on demand Karlsson and Toner (2000) and more recently Fadda et al. (2009). Compared to other methods, freezing to cryogenic temperatures allows long shelf lives and genetic stability (Karlsson and Toner, 2000). Unfortunately, cryopreserved cells are damaged by the cryopreservation process itself (Mazur, 2004). This loss (up to 50 %) can be tolerated for some cell lineages, but it’s unacceptable for others, as the human Mesenchymal Stem Cells (hMSCs) from Umbilical Cord Blood (UCB), whose collection and isolation is known to be difficult (Bieback et al., 2004). In this case, an optimal cryopreservation protocol is mandatory. Due to the high number of trials actually required for experimental optimization, mathematical modelling is considered a practical solution. To this aim, the osmotic properties need to be first estimated in order to determine the volume of residual intra-cellular water left by osmosis to form lethal ice or glass. In this work, the hMSCs from UCB of three different donors, after informed consent, have been isolated by a density gradient centrifugation method. The successful isolation has been verified through phenotypic cytofluorimetric analysis, and adipogenesis/osteogenesis capability differentiations. Osmotic properties, namely inactive cell volume, water and CPA (DMSO) permeabilities, have been determined by means of experimental runs carried out under hypertonic conditions (obtained with the addition of sucrose or DMSO), at three different temperatures. Cells volumes excursions have been measured by a potenziometric device (Coulter Counter) under equilibrium and dynamic conditions. Linear and non-linear regression analyses have been carried out to determine the adjustable parameters by means of the two parameters bi-compartimental model by Kleinahns (1998), as applied to a single-sized cell population (i.e. identical cells with size equal to the average). It is found that, the inactive volume fraction of hMSC from UCB apparently changes (increase) when DMSO is used instead of sucrose, thus limiting cell volume excursion during swelling. It is hypothesized that, a cell volume control system is activated during swelling, probably due to the action of ion pumps.

Modular Extracellular Sensor Architecture for Engineering Mammalian Cell-based Devices

Engineering mammalian cell-based devices that monitor and therapeutically modulate human physiology is a promising and emerging frontier in clinical synthetic biology. However, realizing this vision will require new technologies enabling engineered circuitry to sense and respond to physiologically relevant cues. No existing technology enables an engineered cell to sense exclusively extracellular ligands, including proteins and pathogens, without relying upon native cellular receptors or signal transduction pathways that may be subject to crosstalk with native cellular components. To address this need, we here report a technology we term a Modular Extracellular Sensor Architecture (MESA). This self-contained receptor and signal transduction platform is maximally orthogonal to native cellular processes and comprises independent, tunable protein modules that enable performance optimization and straightforward engineering of novel MESA that recognize novel ligands. We demonstrate ligand-inducible activation of MESA signaling, optimization of receptor performance using design-based approaches, and generation of MESA biosensors that produce outputs in the form of either transcriptional regulation or transcription-independent reconstitution of enzymatic activity. This systematic, quantitative platform characterization provides a framework for engineering MESA to recognize novel ligands and for integrating these sensors into diverse mammalian synthetic biology applications.

The effect of the encapsulation of bacteria in redox phospholipid polymer hydrogels on electron transfer efficiency in living cell-based devices

Development of living cell-based devices holds great promise in many biomedical and industrial applications. To increase our understanding of the process, we investigated the biological and electrochemical properties of a redox phospholipid polymer hydrogel containing an electron-generating bacteria (Shewanella oneidensis MR-1). A water-soluble and amphiphilic phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid-co-vinylferrocene) (PMBVF), was our choice for incorporation into a hydrogel matrix that promotes encapsulation of bacteria and acts as an electron transfer mediator. This hydrogel formed spontaneously and encapsulated Shewanella in three-dimensional structures. Visual analysis showed that the encapsulated Shewanella maintained viability and metabolic activity even after long-term storage. Cyclic voltammetry measurement indicated that the PMBVF/poly(vinyl alcohol) (PMBVF/PVA) hydrogel had stable and high electron transfer efficiency. Amperometric measurement showed that the hydrogel could maintain the electron transfer efficiency even when Shewanella was encapsulated. Thus, the PMBVF/PVA hydrogel not only provides a mild environment for long-term bacterial survival but also maintains electron transfer efficiency from the bacteria to the electrode. We conclude that hydrogel/bacteria hybrid biomaterials, such as PMBVF/PVA/Shewanella, may find application in the fabrication of living cell-based devices.

Feasibility of a porcine oral mucosa equivalent: A preclinical study

Oral tissue engineering aims to treat and fill tissue deficits caused by congenital defects, facial trauma, or malignant lesion surgery, as well as to study the biology of oral mucosa. The Food and Drug Administration (FDA) and the European Medicines Agency (EMA) require a large animal model to evaluate cell-based devices, including tissue-engineered oral mucosa, prior to initiating human clinical studies. Porcine oral mucosa is non-keratinized and resembles that of humans more closely than any other animal in terms of structure and composition; however, there have not been any reports on the reconstruction of a porcine oral mucosa equivalent, probably due to the difficulty to culture porcine fibroblasts. In this study, we demonstrate the feasibility of a 3D porcine oral mucosa equivalent based on a collagen-GAG-chitosan scaffold, as well as reconstructed porcine epithelium by using an amniotic membrane as support, or without any support in form of epithelial cell sheets by using thermoresponsive culture plates. Explants technique was used for the isolation of the porcine fibroblasts and a modified fibroblast medium containing 20% fetal calf serum was used for their culture. The histological and transmission electron microscopic analyses of the resulting porcine oral mucosa models showed the presence of non-keratinized epithelia expressing keratin 13, the major differentiation marker of non-keratinized oral mucosa, in all models, and the presence of newly synthesized collagen fibers in the lamina propria equivalent of the full-thickness model, indicating the functionality of porcine fibroblasts.

Spatial Control of Cell Adhesion and Patterning through Mussel-Inspired Surface Modification by Polydopamine

The spatial control and patterning of mammalian cells were achieved by using the universal adhesive property of mussel-inspired polydopamine (PDA). The self-polymerization of dopamine, a small molecule inspired by the DOPA motif of mussel foot proteins, resulted in the formation of a PDA adlayer when aqueous dopamine solution was continuously injected into poly(dimethylsiloxane) microchannels. We found that various cells (fibrosarcoma HT1080, mouse preosteoblast MC3T3-E1, and mouse fibroblast NIH-3T3) predominantly adhered to PDA-modified regions, maintaining their normal morphologies. The cells aligned in the direction of striped PDA patterns, and this tendency was not limited by the type of cell line. Because PDA modification does not require complex chemical reactions and is applicable to any type of material, it enables cell patterning in a simple and versatile manner as opposed to conventional methods based on the immobilization of adhesive proteins. The PDA-based method of cell patterning should be useful in many biomaterial research areas such as the fabrication of tissue engineering scaffolds, cell-based devices for drug screening, and the fundamental study of cell-material interactions.

Hydrogel cell patterning incorporating photocaged RGDS peptides

As we aim towards enhancing our knowledge of complex cell behaviors and developing intricate cell-based devices and improved therapeutics, it becomes imperative that we be able to control and manipulate the spatial localization of cells. Here we have developed a novel strategy to pattern cells using a hyaluronic acid hydrogel material and photocaged RGDS (Arg-Gly-Asp-Ser) peptides. In this report, we discuss the chemical synthesis and photoactive properties of the caged peptides as well as the subsequent binding of these peptides to our hydrogel base. We further demonstrate the ability of this modified hydrogel material to pattern fibroblast cells on the micron scale using near-UV light exposure through a patterned photomask to selectively switch areas of the hydrogel surface from cell non-adhesive to cell adhesive. The cells are found to adhere and proliferate along the developed line patterns for at least 2.5 days, demonstrating significantly enhanced pattern longevity in comparison with previously reported studies.

A Microfluidic Manipulator for Enrichment and Alignment of Moving Cells and Particles

Grooved structures have been widely studied in particle separation and fluid mixing in microfluidic channel systems. In this brief report, we demonstrate the use of patterning flows produced by two different sorts of grooved surfaces: single slanted groove series (for enrichment patterns) and V-shaped groove series (for focusing patterns), into a microfluidic device to continuously manipulate the flowing particles, including microbeads with 6 microm, 10 microm, and 20 microm in diameter and mouse dendritic cells of comparable sizes to the depth of the channel. The device with grooved channels was developed and fabricated by soft-lithographic techniques. The particle distributions after passing through the single slanted grooves illustrate the size-dependent enrichment profiles. On the other hand, particles passing through the V-shaped grooves show focusing patterns downstream, for the combination effect from both sides of single slanted grooves setup side-by-side. Compared with devices utilizing sheath flows, the focusing patterns generated in this report are unique without introducing additional flow control. The alignment of the concentrated particles is expected to facilitate the visualization of sizing and counting in cell-based devices. On the other hand, the size-dependent patterns of particle distributions have the potential for the application of size-based separation.

Optimization of preservation conditions of As (III) bioreporter bacteria

Long-term preservation of bioreporter bacteria is essential for the functioning of cell-based detection devices, particularly when field application, e.g., in developing countries, is intended. We varied the culture conditions (i.e., the NaCl content of the medium), storage protection media, and preservation methods (vacuum drying vs. encapsulation gels remaining hydrated) in order to achieve optimal preservation of the activity of As (III) bioreporter bacteria during up to 12 weeks of storage at 4 degrees C. The presence of 2% sodium chloride during the cultivation improved the response intensity of some bioreporters upon reconstitution, particularly of those that had been dried and stored in the presence of sucrose or trehalose and 10% gelatin. The most satisfying, stable response to arsenite after 12 weeks storage was obtained with cells that had been dried in the presence of 34% trehalose and 1.5% polyvinylpyrrolidone. Amendments of peptone, meat extract, sodium ascorbate, and sodium glutamate preserved the bioreporter activity only for the first 2 weeks, but not during long-term storage. Only short-term stability was also achieved when bioreporter bacteria were encapsulated in gels remaining hydrated during storage.

Electrochemical impedance spectroscopy to study physiological changes affecting the red blood cell after invasion by malaria parasites

The malaria parasite, Plasmodium falciparum, invades human erythrocytes and induces dramatic changes in the host cell. The idea of this work was to use RBC modified electrode to perform electrochemical impedance spectroscopy (EIS) with the aim of monitoring physiological changes affecting the erythrocyte after invasion by the malaria parasite. Impedance cell-based devices are potentially useful to give insight into cellular behavior and to detect morphological changes. The modelling of impedance plots (Nyquist diagram) in equivalent circuit taking into account the presence of the cellular layer, allowed us pointing out specific events associated with the development of the parasite such as (i) strong changes in the host cell cytoplasm illustrated by changes in the film capacity, (ii) perturbation of the ionic composition of the host cell illustrated by changes in the film resistance, (iii) releasing of reducer (lactic acid or heme) and an enhanced oxygen consumption characterized by changes in the charge transfer resistance and in the Warburg coefficient characteristic of the redox species diffusion. These results show that the RBC-based device may help to analyze strategic events in the malaria parasite development constituting a new tool in antimalarial research.

Particle enrichment employing grooved microfluidic channels

The well-studied chaotic micromixer has found its application on the enrichment of microparticles. Here, we report the use of such patterning flows produced by a grooved surface integrated into a microfluidic device to continuously concentrate the flowing particles of comparable sizes to the depth of the channel. The particle distributions after passing through the grooves illustrate the enrichment profiles and the size-dependent patterns. We expect that the alignment of the concentrated particles can facilitate the visualization of sizing and counting in cell-based devices.

Assembly of polystyrene microspheres and its application in cell micropatterning

Cell micropatterning has important applications in the development of biosensors and lab-on-a-chip devices, tissue engineering and fundamental cell biology studies. The conventional micropatterning techniques involve patterning of cells over a planar substrate. In this paper, we propose the introduction of topographical features on cell adhesive regions to enhance cell adhesion and function. The textured surface is created by assembly of polystyrene microspheres and the topographical parameters can be varied systematically by changing the size and density of the particles. A technique of generating spatial arrangement of microspheres on a nonfouling background is developed. This is achieved by using a bi-functional template which has a patterned hydrophobic parylene film to facilitate self-assembly of particles; after assembly, the film is liftoff, revealing a cell resistance background which is compatible with cell micropatterning. Particles were assembled by selective wetting of the hydrophobic–hydrophilic template. A fluidic chamber was designed to control the movement of the particle suspension across the template so as to attain uniform particle array over large area. This method of cell micropatterning can improve the efficiency and functionality of cell-based devices. It can also be used for examining the effects of topographical cues on cell–substrate adhesion which can provide valuable insights into cell biology and design of biomaterials.

A TAD Further: Exogenous Control of Gene Activation

Designer molecules that can be used to impose exogenous control on gene transcription, artificial transcription factors (ATFs), are highly desirable as mechanistic probes of gene regulation, as potential therapeutic agents, and as components of cell-based devices. Recently, several advances have been made in the design of ATFs that activate gene transcription (activator ATFs), including reports of small-molecule-based systems and ATFs that exhibit potent activity. However, the many open mechanistic questions about transcriptional activators, in particular, the structure and function of the transcriptional activation domain (TAD), have hindered rapid development of synthetic ATFs. A compelling need thus exists for chemical tools and insights toward a more detailed portrait of the dynamic process of gene activation.

Micromolding of photocrosslinkable hyaluronic acid for cell encapsulation and entrapment

Micropatterning of hydrogels is potentially useful for a variety of applications, including tissue engineering, fundamental biological studies, diagnostics, and high-throughput screening. Although synthetic polymers have been developed for these applications, natural polymers such as polysaccharides may have advantages for biological samples and cell-based devices because they are natural components of the in vivo microenvironment. In this study, we synthesized and used hyaluronic acid (HA) modified with photoreactive methacrylates to fabricate microstructures as functional components of microfabricated devices. To demonstrate the universality of this approach, two types of microstructures were formed. In the first approach, HA microstructures were fabricated and used as docking templates to enable the subsequent formation of cell microarrays within low shear stress regions of the patterns. Cells within these microwells remained viable, could generate spheroids, and could be retrieved using mechanical disruption. In the second approach, cells were encapsulated directly within the HA hydrogels. Arrays of viable embryonic stem (ES) cells or fibroblasts were encapsulated within HA hydrogels and could later be recovered using enzymatic digestion of the microstructures. These results demonstrate that it is possible to incorporate photocrosslinkable HA, a natural, versatile, degradable, and biocompatible biopolymer, into micro-electromechanical systems.

Engineering a biospecific communication pathway between cells and electrodes

Methods for transducing the cellular activities of mammalian cells into measurable electronic signals are important in many biotechnical applications, including biosensors, cell arrays, and other cell-based devices. This manuscript describes an approach for functionally integrating cellular activities and electrical processes in an underlying substrate. The cells are engineered with a cell-surface chimeric receptor that presents the nonmammalian enzyme cutinase. Action of this cell-surface cutinase on enzyme substrate self-assembled monolayers switches a nonelectroactive hydroxyphenyl ester to an electroactive hydroquinone, providing an electrical activity that can be identified with cyclic voltammetry. In this way, cell-surface enzymatic activity is transduced into electronic signals. The development of strategies to directly interface the activities of cells with materials will be important to enabling a broad class of hybrid microsystems that combine living and nonliving components.