Imaging approaches for monitoring three-dimensional cell and tissue culture systems.

The past decade has seen an increasing demand for more complex, reproducible and physiologically relevant tissue cultures that can mimic the structural and biological features of living tissues. Monitoring the viability, development and responses of such tissues in real-time are challenging due to the complexities of cell culture physical characteristics and the environments in which these cultures need to be maintained in. Significant developments in optics, such as optical manipulation, improved detection and data analysis, have made optical imaging a preferred choice for many three-dimensional (3D) cell culture monitoring applications. The aim of this review is to discuss the challenges associated with imaging and monitoring 3D tissues and cell culture, and highlight topical label-free imaging tools that enable bioengineers and biophysicists to non-invasively characterise engineered living tissues.

Characterising a Custom-Built Radio Frequency PECVD Reactor to Vary the Mechanical Properties of TMDSO Films.

Plasma-polymerised tetramethyldisiloxane (TMDSO) films are frequently applied as coatings for their abrasion resistance and barrier properties. By manipulating the deposition parameters, the chemical structure and thus mechanical properties of the films can also be controlled. These mechanical properties make them attractive as energy adsorbing layers for a range of applications, including carbon fibre composites. In this study, a new radio frequency (RF) plasma-enhanced chemical vapour deposition (PECVD) plasma reactor was designed with the capability to coat fibres with an energy adsorbing film. A key characterisation step for the system was establishing how the properties of the TMDSO films could be modified and compared with those deposited using a well-characterized microwave (MW) PECVD reactor. Film thickness and chemistry were determined with ellipsometry and X-ray photoelectron spectroscopy, respectively. The mechanical properties were investigated by nanoindentation and atomic force microscopy with peak-force quantitative nanomechanical mapping. The RF PECVD films had a greater range of Young's modulus and hardness values than the MW PECVD films, with values as high as 56.4 GPa and 7.5 GPa, respectively. These results demonstrated the varied properties of TMDSO films that could in turn be deposited onto carbon fibres using a custom-built RF PECVD reactor.

Extending In-Plane Impedance Measurements from 2D to 3D Cultures: Design Considerations.

Three-dimensional (3D) cell cultures have recently emerged as tools for biologically modelling the human body. As 3D models make their way into laboratories there is a need to develop characterisation techniques that are sensitive enough to monitor the cells in real time and without the need for chemical labels. Impedance spectroscopy has been shown to address both of these challenges, but there has been little research into the full impedance spectrum and how the different components of the system affect the impedance signal. Here we investigate the impedance of human fibroblast cells in 2D and 3D collagen gel cultures across a broad range of frequencies (10 Hz to 5 MHz) using a commercial well with in-plane electrodes. At low frequencies in both 2D and 3D models it was observed that protein adsorption influences the magnitude of the impedance for the cell-free samples. This effect was eliminated once cells were introduced to the systems. Cell proliferation could be monitored in 2D at intermediate frequencies (30 kHz). However, the in-plane electrodes were unable to detect any changes in the impedance at any frequency when the cells were cultured in the 3D collagen gel. The results suggest that in designing impedance measurement devices, both the nature and distribution of the cells within the 3D culture as well as the architecture of the electrodes are key variables.

Three-Dimensional (3D) cell culture monitoring: Opportunities and challenges for impedance spectroscopy.

Three-dimensional (3D) cell culture has developed rapidly over the past 5-10 years with the goal of better replicating human physiology and tissue complexity in the laboratory. Quantifying cellular responses is fundamental in understanding how cells and tissues respond during their growth cycle and in response to external stimuli. There is a need to develop and validate tools that can give insight into cell number, viability, and distribution in real-time, nondestructively and without the use of stains or other labelling processes. Impedance spectroscopy can address all of these challenges and is currently used both commercially and in academic laboratories to measure cellular processes in 2D cell culture systems. However, its use in 3D cultures is not straight forward due to the complexity of the electrical circuit model of 3D tissues. In addition, there are challenges in the design and integration of electrodes within 3D cell culture systems. Researchers have used a range of strategies to implement impedance spectroscopy in 3D systems. This review examines electrode design, integration, and outcomes of a range of impedance spectroscopy studies and multiparametric systems relevant to 3D cell cultures. While these systems provide whole culture data, impedance tomography approaches have shown how this technique can be used to achieve spatial resolution. This review demonstrates how impedance spectroscopy and tomography can be used to provide real-time sensing in 3D cell cultures, but challenges remain in integrating electrodes without affecting cell culture functionality. If these challenges can be addressed and more realistic electrical models for 3D tissues developed, the implementation of impedance-based systems will be able to provide real-time, quantitative tracking of 3D cell culture systems.

Controlled release from PCL-alginate microspheres via secondary encapsulation using GelMA/HAMA hydrogel scaffolds.

Controlling the release of bioactive agents has important potential applications in tissue engineering. While microspheres have been investigated to manipulate release rates, the majority of these investigations have been based on delivery into aqueous media, whereas the cellular environment in tissue engineering is more typically a hydrogel scaffold. If drug-loaded microspheres are introduced within scaffolds to deliver biologically active substances in situ, it is crucial to understand how the release rate is influenced by interactions between the microspheres and the scaffold. Here, we report the fabrication and characterization of a biodegradable scaffold that contains composite microspheres and is suitable for biological applications. Our approach evaluates the influence on the release profile of a model drug (FITC-dextran sulfate) from alginate and PCL-alginate microspheres within a hydrogel construct forming a secondary encapsulation. Increasing the degree of crosslinking in the secondary encapsulation matrix led to a slower cumulative release from 36% to 15%, from the alginate microspheres, whereas a decrease from 26% to 6% was observed for the PCL-alginate microspheres. These results suggest that the release of bioactive molecules can be fine tuned by independently engineering the properties of the scaffold and microspheres.

Fabrication of a Biocompatible Liquid Crystal Graphene Oxide-Gold Nanorods Electro- and Photoactive Interface for Cell Stimulation.

For decades, electrode-tissue interfaces are pursued to establish electrical stimulation as a reliable means to control neuronal cells behavior. However, spreading of electrical currents in tissues limits its spatial precision. Thus, optical cues, such as near-infrared (NIR) light, are explored as alternatives. Presently, NIR stimulation requires higher energy input than electrical methods despite introduction of light absorbers, e.g., gold nanoparticles. As potential solution, NIR and electrical costimulation are proposed but with limited interfaces capable of sustaining this stimulation technique. Here, a novel electroactive nanocomposite with photoactive properties in the NIR range is constructed by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride/N-hydroxysulfosuccinimide sodium (EDC)/NHS conjugation of liquid crystal graphene oxide (LCGO) to protein-coated gold nanorods (AuNR). The liquid crystal graphene oxide-gold nanorod nanocomposite (LCGO-AuNR) is fabricated into a hydrophilic electrode-coating via drop-casting, making it appropriate for versatile electrode-tissue interface fabrication. UV-vis spectrophotometry results demonstrate that LCGO-AuNR presents an absorbance peak at 798 nm (NIR range). Cyclic voltammetry measurements further confirm its electroactive capacitive properties. Furthermore, LCGO-AuNR coating supports cell adhesion, proliferation, and differentiation of NG108-15 neuronal cells. This biocompatible interface is anticipated, with ideal electrical and optical properties for NIR and electrical costimulation, to enable further development of the technique for energy-efficient and precise neuronal cell modulation.

Multitechnique investigation into the aqueous behavior of plasma polymers.

Plasma polymers are often used in applications requiring aqueous immersion; therefore, it is important to understand how this exposure affects the physical and chemical properties of the films. Three different plasma polymer films were deposited at different distances from the electrode, and the film properties were characterized using contact angle, ellipsometry, and x-ray photoelectron spectroscopy. The film behaviors in aqueous solutions were studied via quartz crystal microbalance with dissipation (QCM-D). Exposure to buffer solutions produced significant swelling of the plasma polymerized acrylic acid films, with swelling increasing with distance from the powered electrode, results that could be correlated with changes in film chemistry. Plasma polymerized octadiene and allylamine exhibited little swelling. These films exhibited changes in thickness and contact angle with respect to distance from the electrode, but this had little influence on their behavior in aqueous solution. By combining QCM-D with the more traditional surface chemical analysis techniques, the authors have been able to explore both swelling behavior and the effect that sample position and thus deposition parameters have on film properties and aqueous behavior. This approach gives the authors the basis to define deposition parameters to assist the engineering of thin films for applications such as biosensing and tissue engineering applications where specific chemistries and film behaviors are desired.

Toward Immunocompetent 3D Skin Models.

3D human skin models provide a platform for toxicity testing, biomaterials evaluation, and investigation of fundamental biological processes. However, the majority of current in vitro models lack an inflammatory system, vasculature, and other characteristics of native skin, indicating scope for more physiologically complex models. Looking at the immune system, there are a variety of cells that could be integrated to create novel skin models, but to do this effectively it is also necessary to understand the interface between skin biology and tissue engineering as well as the different roles the immune system plays in specific health and disease states. Here, a progress report on skin immunity and current immunocompetent skin models with a focus on construction methods is presented; scaffold and cell choice as well as the requirements of physiologically relevant models are elaborated. The wide range of technological and fundamental challenges that need to be addressed to successfully generate immunocompetent skin models and the steps currently being made globally by researchers as they develop new models are explored. Induced pluripotent stem cells, microfluidic platforms to control the model environment, and new real-time monitoring techniques capable of probing biochemical processes within the models are discussed.

pH-dependent lipid vesicle interactions with plasma polymerized thin films.

Model lipid vesicle and supported lipid bilayer (SLB) systems are used in a variety of applications including biosensing, cell membrane mimics, and drug delivery. Exposure of a surface to a vesicle solution provides a straightforward method for creating such systems via vesicle adsorption and collapse. However, this process is complex and the relationship between the surface physicochemical properties and vesicle collapse is poorly understood. Plasma polymers are thin conformal films that can be applied to a variety of materials to modify surface properties. This paper uses quartz crystal microbalance with dissipation and fluorescence recovery after photobleaching (FRAP) to explore lipid vesicle interactions with plasma polymerized acrylic acid (ppAAc), allylamine (ppAAm), and ppAAc/ppAAm micropatterns. Vesicle interactions were dependent on plasma polymer chemistry and pH of the buffer solution. Vesicles readily and stably adsorbed to ppAAm over a wide pH range. ppAAc demonstrated limited interactions at pH 7 and vesicle adsorption at pH 4. Vesicle collapse and SLB formation could be induced using a pH change. FRAP was used to explore the fluidity of the lipid structures on both the patterned and unpatterned plasma polymer films. On ppAAm/ppAAc micropatterns, pH transitions combined with the presence of chemically distinct regions on the same substrate enabled immobile lipid islands on ppAAc to be surrounded by fluid lipid regions on ppAAm. This work demonstrates that plasma polymer films could enable spatially controlled vesicle adsorption and SLB formation on a wide variety of different substrates.

Ultrasonically enhanced fractionation of milk fat in a litre-scale prototype vessel.

The ultrasonic fractionation of milk fat in whole milk to fractions with distinct particle size distributions was demonstrated using a stage-based ultrasound-enhanced gravity separation protocol. Firstly, a single stage ultrasound gravity separation was characterised after various sonication durations (5-20 min) with a mass balance, where defined volume partitions were removed across the height of the separation vessel to determine the fat content and size distribution of fat droplets. Subsequent trials using ultrasound-enhanced gravity separation were carried out in three consecutive stages. Each stage consisted of 5 min sonication, with single and dual transducer configurations at 1 MHz and 2 MHz, followed by aliquot collection for particle size characterisation of the formed layers located at the bottom and top of the vessel. After each sonication stage, gentle removal of the separated fat layer located at the top was performed. Results indicated that ultrasound promoted the formation of a gradient of vertically increasing fat concentration and particle size across the height of the separation vessel, which became more pronounced with extended sonication time. Ultrasound-enhanced fractionation provided fat enriched fractions located at the top of the vessel of up to 13 ± 1% (w/v) with larger globules present in the particle size distributions. In contrast, semi-skim milk fractions located at the bottom of the vessel as low as 1.2 ± 0.01% (w/v) could be produced, containing proportionally smaller sized fat globules. Particle size differentiation was enhanced at higher ultrasound energy input (up to 347 W/L). In particular, dual transducer after three-stage operation at maximum energy input provided highest mean particle size differentiation with up to 0.9 µm reduction in the semi-skim fractions. Higher frequency ultrasound at 2 MHz was more effective in manipulating smaller sized fat globules retained in the later stages of skimming than 1 MHz. While 2 MHz ultrasound removed 59 ± 2% of the fat contained in the initial sample, only 47 ± 2% was removed with 1 MHz after 3 ultrasound-assisted fractionation stages.

Bacterial response to different surface chemistries fabricated by plasma polymerization on electrospun nanofibers.

Control over bacterial attachment and proliferation onto nanofibrous materials constitutes a major challenge for a variety of applications, including filtration membranes, protective clothing, wound dressings, and tissue engineering scaffolds. To develop effective devices, the interactions that occur between bacteria and nanofibers with different morphological and physicochemical properties need to be investigated. This paper explores the influence of fiber surface chemistry on bacterial behavior. Different chemical functionalities were generated on the surface of electrospun polystyrene nanofibers through plasma polymerization of four monomers (acrylic acid, allylamine, 1,7-octadiene, and 1,8-cineole). The interactions of Escherichia coli with the surface modified fibers were investigated through a combination of scanning electron microscopy and confocal laser scanning microscopy. Fiber wettability, surface charge, and chemistry were found to affect the ability of bacterial cells to attach and proliferate throughout the nanofiber meshes. The highest proportion of viable cells attachment occurred on the hydrophilic amine rich coating, followed by the hydrophobic octadiene. The acrylic acid coating rich in carboxyl groups showed a significantly lower attraction of bacterial cells. The 1,8-cineole retained the antibacterial activity of the monomer, resulting with a high proportion of dead isolated cells attached onto the fibers. Results showed that the surface chemistry properties of nanofibrous membranes can be strategically tuned to control bacterial behavior.

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells.

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

Versatile SERS sensing based on black silicon.

Black Si (b-Si) with gold or silver metal coating has been shown to be an extremely effective substrate for surface-enhanced Raman scattering (SERS). Here, we demonstrate that it is also a highly versatile SERS platform, as it supports a wide range of surface functionalizations. In particular, we report the use of a molecularly imprinted polymer (MIP) coating and a hydrophobic coating on b-Si to establish two different sensing modalities. First, using a MIP layer on Au-coated b-Si, we show selective sensing of two closely related varieties of tetracycline. Second, a hydrophobic coating was used to concentrate the analyte adsorbed on gold colloidal nanoparticles, thus increasing the sensitivity of the measurement by an order of magnitude. In this experiment, Au nanoparticles and analyte were mixed just before SERS measurements and were concentrated by drop-drying on the super-hydrophobic b-Si. These approaches are promising for SERS measurements that are sensitive to the aging of bare plasmonic metal-coated substrates.

Electrospun polystyrene fiber diameter influencing bacterial attachment, proliferation, and growth.

Electrospun materials have been widely investigated in the past few decades as candidates for tissue engineering applications. However, there is little available data on the mechanisms of interaction of bacteria with electrospun wound dressings of different morphology and surface chemistry. This knowledge could allow the development of effective devices against bacterial infections in chronic wounds. In this paper, the interactions of three bacterial species (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) with electrospun polystyrene meshes were investigated. Bacterial response to meshes with different fiber diameters was assessed through a combination of scanning electron microscopy (SEM) and confocal microscopy. Experiments included attachment studies in liquid medium but also directly onto agar plates; the latter was aimed at mimicking a chronic wound environment. Fiber diameter was shown to affect the ability of bacteria to proliferate within the fibrous networks, depending on cell size and shape. The highest proliferation rates occurred when fiber diameter was close to the bacterial size. Nanofibers were found to induce conformational changes of rod shaped bacteria, limiting the colonization process and inducing cell death. The data suggest that simply tuning the morphological properties of electrospun fibers may be one strategy used to control biofilm formation within wound dressings.

Fabrication of a platform to isolate the influences of surface nanotopography from chemistry on bacterial attachment and growth.

Billions of dollars are spent annually worldwide to combat the adverse effects of bacterial attachment and biofilm formation in industries as varied as maritime, food, and health. While advances in the fabrication of antifouling surfaces have been reported recently, a number of the essential aspects responsible for the formation of biofilms remain unresolved, including the important initial stages of bacterial attachment to a substrate surface. The reduction of bacterial attachment to surfaces is a key concept in the prevention or minimization of biofilm formation. The chemical and physical characteristics of both the substrate and bacteria are important in understanding the attachment process, but substrate modification is likely the most practical route to enable the extent of bacterial attachment taking place to be effectively controlled. The microtopography and chemistry of the surface are known to influence bacterial attachment. The role of surface chemistry versus nanotopography and their interplay, however, remain unclear. Most methods used for imparting nanotopographical patterns onto a surface also induce changes in the surface chemistry and vice versa. In this study, the authors combine colloidal lithography and plasma polymerization to fabricate homogeneous, reproducible, and periodic nanotopographies with a controllable surface chemistry. The attachment of Escherichia coli bacteria onto carboxyl (plasma polymerized acrylic acid, ppAAc) and hydrocarbon (plasma polymerized octadiene, ppOct) rich plasma polymer films on either flat or colloidal array surfaces revealed that the surface chemistry plays a critical role in bacterial attachment, whereas the effect of surface nanotopography on the bacterial attachment appears to be more difficult to define. This platform represents a promising approach to allow a greater understanding of the role that surface chemistry and nanotopography play on bacterial attachment and the subsequent biofouling of the surface.

Gold-nanorod-assisted near-infrared stimulation of primary auditory neurons.

Infrared stimulation offers an alternative to electrical stimulation of neuronal tissue, with potential for direct, non-contact activation at high spatial resolution. Conventional methods of infrared neural stimulation (INS) rely on transient heating due to the absorption of relatively intense laser beams by water in the tissue. However, the water absorption also limits the depth of penetration of light in tissue. Therefore, the use of a near-infrared laser at 780 nm to stimulate cultured rat primary auditory neurons that are incubated with silica-coated gold nanorods (Au NRs) as an extrinsic absorber is investigated. The laser-induced electrical behavior of the neurons is observed using whole-cell patch clamp electrophysiology. The nanorod-treated auditory neurons (NR-ANs) show a significant increase in electrical activity compared with neurons that are incubated with non-absorbing silica-coated gold nanospheres and control neurons with no gold nanoparticles. The laser-induced heating by the nanorods is confirmed by measuring the transient temperature increase near the surface of the NR-ANs with an open pipette electrode. These findings demonstrate the potential to improve the efficiency and increase the penetration depth of INS by labeling nerves with Au NRs and then exposing them to infrared wavelengths in the water window of tissue.