Non-invasive methods of monitoring Fe3O4 magnetic nanoparticle toxicity in human liver HepaRG cells using impedance biosensing and Coherent anti-Stokes Raman spectroscopic (CARS) microscopy.

Functionalized nanoparticles have been developed for use in nanomedicines for treating life threatening diseases including various cancers. To ensure safe use of these new nanoscale reagents, various assays for biocompatibility or cytotoxicity in vitro using cell lines often serve as preliminary assessments prior to in vivo animal testing. However, many of these assays were designed for soluble, colourless materials and may not be suitable for coloured, non-transparent nanoparticles. Moreover, cell lines are not always representative of mammalian organs in vivo. In this work, we use non-invasive impedance sensing methods with organotypic human liver HepaRG cells as a model to test the toxicity of PEG-Fe3O4 magnetic nanoparticles. We also use Coherent anti-Stokes Raman Spectroscopic (CARS) microscopy to monitor the formation of lipid droplets as a parameter to the adverse effect on the HepaRG cell model. The results were also compared with two commercial testing kits (PrestoBlue and ATP) for cytotoxicity. The results suggested that the HepaRG cell model can be a more realistic model than commercial cell lines while use of impedance monitoring of Fe3O4 nanoparticles circumventing the uncertainties due to colour assays. These methods can play important roles for scientists driving towards the 3Rs principle - Replacement, Reduction and Refinement.

Debiased ambient vibrations optical coherence elastography to profile cell, organoid and tissue mechanical properties.

The role of the mechanical environment in defining tissue function, development and growth has been shown to be fundamental. Assessment of the changes in stiffness of tissue matrices at multiple scales has relied mostly on invasive and often specialist equipment such as AFM or mechanical testing devices poorly suited to the cell culture workflow.In this paper, we have developed a unbiased passive optical coherence elastography method, exploiting ambient vibrations in the sample that enables real-time noninvasive quantitative profiling of cells and tissues. We demonstrate a robust method that decouples optical scattering and mechanical properties by actively compensating for scattering associated noise bias and reducing variance. The efficiency for the method to retrieve ground truth is validated in silico and in vitro, and exemplified for key applications such as time course mechanical profiling of bone and cartilage spheroids, tissue engineering cancer models, tissue repair models and single cell. Our method is readily implementable with any commercial optical coherence tomography system without any hardware modifications, and thus offers a breakthrough in on-line tissue mechanical assessment of spatial mechanical properties for organoids, soft tissues and tissue engineering.

Quantitative action spectroscopy reveals ARPE19 sensitivity to ultraviolet radiation at 350 nm and 380 nm.

The role of ultraviolet radiation (UVR) exposure in the aetiology of retinal degeneration has been debated for decades with epidemiological evidence failing to find a clear consensus for or against it playing a role. A key reason for this is a lack of foundational research into the response of living retinal tissue to UVR in regard to modern ageing-specific parameters of tissue function. We therefore explored the response of cultured retinal pigmented epithelium (RPE), the loss of which heralds advanced visual decline, to specific wavelengths of UVR across the UV-B and UV-A bands found in natural sunlight. Using a bespoke in vitro UVR exposure apparatus coupled with bandpass filters we exposed the immortalised RPE cell line, ARPE-19, to 10 nm bands of UVR between 290 and 405 nm. Physical cell dynamics were assessed during exposure in cells cultured upon specialist electrode culture plates which allow for continuous, non-invasive electrostatic interrogation of key cell parameters during exposure such as monolayer coverage and tight-junction integrity. UVR exposures were also utilised to quantify wavelength-specific effects using a rapid cell viability assay and a phenotypic profiling assay which was leveraged to simultaneously quantify intracellular reactive oxygen species (ROS), nuclear morphology, mitochondrial stress, epithelial integrity and cell viability as part of a phenotypic profiling approach to quantifying the effects of UVR. Electrical impedance assessment revealed unforeseen detrimental effects of UV-A, beginning at 350 nm, alongside previously demonstrated UV-B impacts. Cell viability analysis also highlighted increased effects at 350 nm as well as 380 nm. Effects at 350 nm were further substantiated by high content image analysis which highlighted increased mitochondrial dysfunction and oxidative stress. We conclude that ARPE-19 cells exhibit a previously uncharacterised sensitivity to UV-A radiation, specifically at 350 nm and somewhat less at 380 nm. If upheld in vivo, such sensitivity will have impacts upon geoepidemiological risk scoring of macular sensitivity.

Optical Cellular Micromotion: A New Paradigm to Measure Tumor Cells Invasion within Gels Mimicking the 3D Tumor Environments.

Measuring tumor cell invasiveness through 3D tissues, particularly at the single-cell level, can provide important mechanistic understanding and assist in identifying therapeutic targets of tumor invasion. However, current experimental approaches, including standard in vitro invasion assays, have limited physiological relevance and offer insufficient insight into the vast heterogeneity in tumor cell migration through tissues. To address these issues, here the concept of optical cellular micromotion is reported on, where digital holographic microscopy is used to map the optical nano- to submicrometer thickness fluctuations within single-cells. These fluctuations are driven by the dynamic movement of subcellular structures including the cytoskeleton and inherently associated with the biological processes involved in cell invasion within tissues. It is experimentally demonstrated that the optical cellular micromotion correlates with tumor cells motility and invasiveness both at the population and single-cell levels. In addition, the optical cellular micromotion significantly reduced upon treatment with migrastatic drugs that inhibit tumor cell invasion. These results demonstrate that micromotion measurements can rapidly and non-invasively determine the invasive behavior of single tumor cells within tissues, yielding a new and powerful tool to assess the efficacy of approaches targeting tumor cell invasiveness.

Impedance-Optical Dual-Modal Cell Culture Imaging With Learning-Based Information Fusion.

While Electrical Impedance Tomography (EIT) has found many biomedicine applications, better image quality is needed to provide quantitative analysis for tissue engineering and regenerative medicine. This paper reports an impedance-optical dual-modal imaging framework that primarily targets at high-quality 3D cell culture imaging and can be extended to other tissue engineering applications. The framework comprises three components, i.e., an impedance-optical dual-modal sensor, the guidance image processing algorithm, and a deep learning model named multi-scale feature cross fusion network (MSFCF-Net) for information fusion. The MSFCF-Net has two inputs, i.e., the EIT measurement and a binary mask image generated by the guidance image processing algorithm, whose input is an RGB microscopic image. The network then effectively fuses the information from the two different imaging modalities and generates the final conductivity image. We assess the performance of the proposed dual-modal framework by numerical simulation and MCF-7 cell imaging experiments. The results show that the proposed method could improve the image quality notably, indicating that impedance-optical joint imaging has the potential to reveal the structural and functional information of tissue-level targets simultaneously.

Application of Impedance-Based Techniques in Hepatology Research.

There are a variety of end-point assays and techniques available to monitor hepatic cell cultures and study toxicity within in vitro models. These commonly focus on one aspect of cell metabolism and are often destructive to cells. Impedance-based cellular assays (IBCAs) assess biological functions of cell populations in real-time by measuring electrical impedance, which is the resistance to alternating current caused by the dielectric properties of proliferating of cells. While the uses of IBCA have been widely reported for a number of tissues, specific uses in the study of hepatic cell cultures have not been reported to date. IBCA monitors cellular behaviour throughout experimentation non-invasively without labelling or damage to cell cultures. The data extrapolated from IBCA can be correlated to biological events happening within the cell and therefore may inform drug toxicity studies or other applications within hepatic research. Because tight junctions comprise the blood/biliary barrier in hepatocytes, there are major consequences when these junctions are disrupted, as many pathologies centre around the bile canaliculi and flow of bile out of the liver. The application of IBCA in hepatology provides a unique opportunity to assess cellular polarity and patency of tight junctions, vital to maintaining normal hepatic function. Here, we describe how IBCAs have been applied to measuring the effect of viral infection, drug toxicity /IC50, cholangiopathies, cancer metastasis and monitoring of the gut-liver axis. We also highlight key areas of research where IBCAs could be used in future applications within the field of hepatology.

Blue-light induced breakdown of barrier function on human retinal epithelial cells is mediated by PKC-ζ over-activation and oxidative stress.

We aimed to study the time course decrease of human retinal pigment epithelium (RPE) barrier function when exposed to blue light. To this end, we cultured ARPE-19 cells on Electrical Cell-substrate Impedance Sensing (ECIS) multi-well arrays. Using an ad hoc light emitting diode (LED) array illumination system together with a set of neutral density filters and a 3-dimensional (3D) printed filter holder, cells were exposed to a gradient of irradiances of blue-light with a measured peak at 468 nm. The electrical resistance between 4 kHz and 64 kHz was recorded during the exposure. Blue light exposure induced a dose-dependent decrease in the resistances at 4 kHz, however the time course resistance at 64 kHz did not show any decrease before t = 52 h. Quantification of the barrier function using mathematical model integrated in the ECIS software showed that blue-light exposure induced a dose-dependent decrease in the barrier function associated with tight junction formation (P < 0.05). This was confirmed by the immunostaining of the tight-junction associated structural protein, Zonula occludens-1 (ZO-1). The detection of reactive oxygen species by carboxy-H2DCFDA confirmed that the blue light induced dose-dependent decrease in the barrier function is mediated by oxidative stress. On a separate experiment, blue-light exposed ARPE-19 cells were treated with 100 nM Protein Kinase C zeta (PKC-ζ) pseudo substrate inhibitor to identify underlying pathway for blue-light induced damage on the barrier function. The treatment with 100 nM PKC-ζ pseudo substrate inhibitor induced faster recovery of the barrier function compared to no treatment. Altogether our results document that blue LED light exposure decreased RPE barrier function in-vitro in a dose-dependent manner, before any cell death occurred. This damage induced by blue-light on tight junctions is mediated by oxidative stress through PKC-ζ activation. The quantification of the healing effect observed by inhibition of PKC-ζ might lead to development of high throughput wound healing assays through ECIS in the future.

Electrical impedance tomography for real-time and label-free cellular viability assays of 3D tumour spheroids.

There is currently a need to culture cells in 3D to better mimic the behaviour of cells growing in the natural environment. In parallel, this calls for novel technologies to assess cell growth in 3D cell culture. In this study, we demonstrated both in silico and in vitro that cell viability inside large cell spheroids could be monitored in real time and label-free with electrical impedance tomography (EIT). Simulations using a single shell model and the effective media approximation (EMA) method were performed to prove the performance of EIT on spheroid imaging and viability monitoring. Then in vitro experiments were conducted to measure in real time a loss of cell viability in MCF-7 breast cancer spheroids when exposed to Triton X-100 and validate with conventional biochemical assays. It is shown that EIT has a spatial resolution of 1.14% and it could monitor the cell mortality over 20% of a spheroid under laboratory noise level. The reconstructed conductivity images for cell mortality induced by the chemical are clear and match the result in the cellular metabolic viability assay. Furthermore, the image reconstruction speed in the experiment was less than 0.3 seconds. Taken together, the results show the potential of EIT for non-destructive real-time and label-free cellular assays in the miniature sensor, providing physiological information in the applications of 3D drug screening and tissue engineering.

Biosynthesis of magnetic nanoparticles by human mesenchymal stem cells following transfection with the magnetotactic bacterial gene mms6.

The use of stem cells to support tissue repair is facilitated by loading of the therapeutic cells with magnetic nanoparticles (MNPs) enabling magnetic tracking and targeting. Current methods for magnetizing cells use artificial MNPs and have disadvantages of variable uptake, cellular cytotoxicity and loss of nanoparticles on cell division. Here we demonstrate a transgenic approach to magnetize human mesenchymal stem cells (MSCs). MSCs are genetically modified by transfection with the mms6 gene derived from Magnetospirillum magneticum AMB-1, a magnetotactic bacterium that synthesises single-magnetic domain crystals which are incorporated into magnetosomes. Following transfection of MSCs with the mms6 gene there is bio-assimilated synthesis of intracytoplasmic magnetic nanoparticles which can be imaged by MR and which have no deleterious effects on cell proliferation, migration or differentiation. The assimilation of magnetic nanoparticle synthesis into mammalian cells creates a real and compelling, cytocompatible, alternative to exogenous administration of MNPs.

Targeted SERS nanosensors measure physicochemical gradients and free energy changes in live 3D tumor spheroids.

Use of multicellular tumor spheroids (MTS) to investigate therapies has gained impetus because they have potential to mimic factors including zonation, hypoxia and drug-resistance. However, analysis remains difficult and often destroys 3D integrity. Here we report an optical technique using targeted nanosensors that allows in situ 3D mapping of redox potential gradients whilst retaining MTS morphology and function. The magnitude of the redox potential gradient can be quantified as a free energy difference (ΔG) and used as a measurement of MTS viability. We found that by delivering different doses of radiotherapy to MTS we could correlate loss of ΔG with increasing therapeutic dose. In addition, we found that resistance to drug therapy was indicated by an increase in ΔG. This robust and reproducible technique allows interrogation of an in vitro tumor-model's bioenergetic response to therapy, indicating its potential as a tool for therapy development.

Cellular micromotion monitored by long-range surface plasmon resonance with optical fluctuation analysis.

Long-range surface plasmon resonance (LRSPR) is a powerful biosensing technology due to a substantially larger probing depth into the medium and sensitivity, compared with conventional SPR. We demonstrate here that LRSPR can provide sensitive noninvasive measurement of the dynamic fluctuation of adherent cells, often referred to as the cellular micromotion. Proof of concept was achieved using confluent layers of 3T3 fibroblast cells and MDA-MB-231 cancer cells. The slope of the power spectral density (PSD) of the optical fluctuations was calculated to determine the micromotion index, and significant differences were measured between live and fixed cell layers. Furthermore, the performances of LRSPR and conventional surface plasmon resonance (cSPR) were compared with respect to micromotion monitoring. Our study showed that the micromotion index of cells measured by LRSPR sensors was higher than when measured with cSPR, suggesting a higher sensitivity of LRSPR to the micromotion of cells. To investigate further this finding, simulations were conducted to establish the relative sensitivities of LRSPR and cSPR to membrane fluctuations. Increased signal intensity was predicted for LRSPR in comparison to cSPR, suggesting that membrane fluctuations play a significant role in the optical micromotion measured in LRSPR. Analogous to cellular micromotion measured using impedance techniques, LRSPR micromotion has the potential to provide important biological information on the metabolic activity and viability of adherent cells.

Paracrine signalling events in embryonic stem cell renewal mediated by affinity targeted nanoparticles.

Stem cell growth and differentiation is controlled by intrinsic and extrinsic factors. The latter includes growth factors, which are conventionally supplied in vitro in media exchanged daily. Here, we illustrate the use of affinity targeted biodegradable nanoparticles to mediate paracrine stimulation as an alternative approach to sustain the growth and pluripotency of mouse embryonic stem cells. Leukaemia Inhibitory Factor (LIF) was encapsulated in biodegradable nanoparticles and targeted to the cell surface using an antibody to the oligosaccharide antigen SSEA-1. Sustained release of LIF from nanoparticles composed of a solid Poly(lactide-co-glycolic acid) polyester or a hydrogel-based liposomal system, we term Nanolipogel, replenished once after each cell passage, proved as effective as daily replenishment with soluble LIF for maintenance of pluripotency after 5 passages using 10(4)-fold less LIF. Our study constitutes an alternative paradigm for stem cell culture, providing dynamic microenvironmental control of extrinsic bioactive factors benefiting stem cell manufacturing.

Raman spectroscopy and CARS microscopy of stem cells and their derivatives.

The characterisation of stem cells is of vital importance to regenerative medicine. Failure to separate out all stem cells from differentiated cells before therapies can result in teratomas - tumours of multiple cell types. Typically, characterisation is performed in a destructive manner with fluorescent assays. A truly non-invasive method of characterisation would be a major breakthrough in stem cell-based therapies. Raman spectroscopy has revealed that DNA and RNA levels drop when a stem cell differentiates into other cell types, which we link to a change in the relative sizes of the nucleus and cytoplasm. We also used Raman spectroscopy to investigate the biochemistry within an early embryo, or blastocyst, which differs greatly from colonies of embryonic stem cells. Certain cell types that differentiate from stem cells can be identified by directly imaging the biochemistry with CARS microscopy; examples presented are hydroxyapatite - a precursor to bone, and lipids in adipocytes.

Tissue engineering for tendon repair.

Tissue engineering aims to induce tissue self-regeneration in vivo or to produce a functional tissue replacement in vitro to be then implanted in the body. To produce a viable and functional tendon, a uniaxially orientated collagen type I matrix has to be generated. Biochemical and physical factors can potentially alter both the production and the organisation of this matrix, and their combination in a dose- and time-dependent manner is probably the key to in vitro engineered tendons. This review discusses the role of these different factors affecting tenocyte growth in a three-dimensional environment in vivo and in vitro, and underlines the future challenge of tendon tissue engineering.

Towards on-line monitoring of cell growth in microporous scaffolds: Utilization and interpretation of complex permittivity measurements.

Here we demonstrate the ability to characterize microporous scaffolds and evaluate cell concentration variation via the utilization and interpretation of complex permittivity measurements (CP), a direct and nondestructive method. Polymer-based microporous scaffolds are of importance to tissue engineering, particularly in the promotion of cell adhesion, proliferation, and differentiation in predefined shapes. Chitosan gel scaffolds were seeded with increasing concentrations of macrophages to simulate cell growth. Complex permittivity measurements were performed using a dielectric probe and a vector network analyzer over a frequency ranging from 200 MHz to 2 GHz. An effective medium theory was applied to interpret the data obtained; respectively, Looyenga and Maxwell-Wagner-Hanai functions were used to retrieve the porosity and the variation of the cell concentration from the CP measurements. Calculated porosities were in agreement with experimental evaluation-porosity ranged from 81-96%. Changes in cell concentration inside the scaffolds upon injection of differing cell concentrations into the scaffold were detected distinguishably. Variations resulting from the cumulative injection of 400-1800 microL of 10(6) cells/mL solution into the scaffold were monitored. Results suggest that CP measurements in combination with an appropriate effective medium approximation can enable on-line monitoring of cell growth within scaffolds.