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Water is a strong mid-infrared absorber, which has hindered the full exploitation of label-free and non-invasive infrared (IR) spectromicroscopy techniques for the study of living biological samples. To overcome this barrier, many researchers have built sophisticated fluidic chambers or microfluidic chips wherein the depth of the liquid medium in the sample compartment is limited to 10 μm or less. Here we report an innovative and simple way to fabricate plastic devices with infrared transparent view-ports enabling infrared spectromicroscopy of living biological samples; therefore the device is named "IR-Live". Advantages of this approach include lower production costs, a minimal need to access a micro-fabrication facility, and unlimited mass or waste exchange for the living samples surrounding the view-port area. We demonstrate that the low-cost IR-Live in combination with microfluidic perfusion techniques enables long term (>60 h) cell culture, which broadens the capability of IR spectromicroscopy for studying living biological samples. To illustrate this, we first applied the device to study protein and lipid polarity in migrating REF52 fibroblasts by collecting 2-dimensional spectral chemical maps at a micrometer spatial resolution. Then, we demonstrated the suitability of our approach to study dynamic cellular events by collecting a time series of spectral maps of U937 monocytes during the early stage of cell attachment to a bio-compatible surface.

The experimental analysis and modeling of AC losses in transport current regime in applied AC magnetic field is reported.

Recent advances in single-cell studies have revealed the vast transcriptomic heterogeneity of cancer-associated fibroblasts (CAFs), with each subset likely having unique roles in the tumor microenvironment. However, it is still unclear how different CAF subsets should be cultured in vitro to recapitulate their in vivo phenotype. The inherent plasticity of CAFs, or their ability to dynamically change their phenotype in response to different environmental stimuli, makes it highly challenging to induce and maintain a specific CAF state in vitro. In this study, we investigated how cell shape and confinement on two-dimensional culture substrates with different stiffnesses influence CAF transcriptomic profile and phenotype. Using micropatterning of polyacrylamide hydrogels to induce shape- and confinement-dependent changes in cell morphology, we observed that micropatterned CAFs exhibited phenotypic shifts towards more desmoplastic and inflammatory CAF subsets. Additionally, micropatterning enabled control over a range of CAF-specific markers and pathways. Lastly, we report how micropatterned and non-micropatterned CAFs respond differently to anti-cancer drugs, highlighting the importance of phenotype-oriented therapy that considers for CAF plasticity and regulatory networks. Control over CAF morphology offers a unique opportunity to establish highly robust CAF phenotypes in vitro, facilitating deeper understanding of CAF plasticity, heterogeneity, and development of novel therapeutic targets. STATEMENT OF SIGNIFICANCE: Cancer-associated fibroblasts (CAFs) are the dominant stromal cell type in many cancers, and recent studies have revealed that they are highly heterogeneous and comprise several subpopulations. It is still unclear how different subsets of CAFs should be cultured in vitro to recapitulate their in vivo phenotype. In this study, we investigated how cell shape and confinement affect CAF transcriptomic profile and phenotype. We report that micropatterned CAFs resemble desmoplastic and inflammatory CAF subsets observed in vivo and respond differently to anti-cancer drugs as compared to non-patterned CAFs. Control over CAF morphology enables the generation of highly robust CAF phenotypes in vitro, facilitating deeper understanding of CAF plasticity and heterogeneity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Inc. All rights reserved.)

Infrared spectro-microscopy is a powerful technique for analysing chemical maps of cells and tissues for biomedical and clinical applications, yet the strong water absorption in the mid-infrared region is a challenge to overcome, as it overlaps with the spectral fingerprints of biological components. Microfluidic chips offer ultimate control over the water layer thickness and are increasingly used in infrared spectro-microscopy. However, the actual impact of the water layer thickness on the instrument's performance is often left to the experimentalist's intuition and the peculiarities of specific instruments. Aiming to experimentally test the amount of absorption introduced by water with varying layer thicknesses, we fabricated a set of microfluidic devices with three controlled chamber thicknesses, each comprising a simple test pattern made of a well-known photoresist SU-8. We employed two infrared spectro-microscopy methods for measurements. The first method involves using a standard FTIR microscope with a benchtop infrared light source. The second method is a quantum infrared microscopy technique, where infrared imaging is achieved by detecting correlated photons in the visible range. We demonstrated that both methods enable the measurement of the absorption spectrum in the mid-IR region, even in the presence of up to a 30 μm thick water layer on top of a sample pattern. Additionally, the Q-IR technique offers practical advantages over synchrotron-based FTIR, such as reduced complexity, cost, and ease of operation.

Robust coordination of surface and volume changes is critical for cell integrity. Few studies have elucidated the plasma membrane (PM) remodeling events during cell surface and volume alteration, especially regarding PM sensing and its subsequent rearrangements. Here, using fission yeast protoplasts, we reveal a Ca 2+ -dependent mechanism for membrane addition that ensures PM integrity and allows its expansion during acute hypoosmotic cell swelling. We show that MscS-like mechanosensitive channels activated by PM tension control extracellular Ca 2+ influx, which triggers direct lipid transfer at endoplasmic reticulum (ER)-PM contact sites by conserved extended-synaptotagmins and accelerates exocytosis, enabling PM expansion necessary for osmotic equilibrium. Defects in any of these key events result in rapid protoplast rupture upon severe hypotonic shock. Our numerical simulations of hypoosmotic expansion further propose a cellular strategy that combines instantaneous non-vesicular lipid transfer with bulk exocytic membrane delivery to maintain PM integrity for dramatic cell surface/volume adaptation., Competing Interests: Declaration of Interests The authors declare no competing interests.

Human neuromuscular diseases represent a diverse group of disorders with unmet clinical need, ranging from muscular dystrophies, such as Duchenne muscular dystrophy (DMD), to neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). In many of these conditions, axonal and neuromuscular synapse dysfunction have been implicated as crucial pathological events, highlighting the need for in vitro disease models that accurately recapitulate these aspects of human neuromuscular physiology. The protocol reported here describes the co-culture of neural spheroids composed of human pluripotent stem cell (PSC)-derived motor neurons and astrocytes, and human PSC-derived myofibers in 3D compartmentalised microdevices to generate functional human neuromuscular circuits in vitro. In this microphysiological model, motor axons project from a central nervous system (CNS)-like compartment along microchannels to innervate skeletal myofibers plated in a separate muscle compartment. This mimics the spatial organization of neuromuscular circuits in vivo. Optogenetics, particle image velocimetry (PIV) analysis, and immunocytochemistry are used to control, record, and quantify functional neuromuscular transmission, axonal outgrowth, and neuromuscular synapse number and morphology. This approach has been applied to study disease-specific phenotypes for DMD and ALS by incorporating patient-derived and CRISPR-corrected human PSC-derived motor neurons and skeletal myogenic progenitors into the model, as well as testing candidate drugs for rescuing pathological phenotypes. The main advantages of this approach are: i) its simple design; ii) the in vivo-like anatomical separation between CNS and peripheral muscle; and iii) the amenability of the approach to high power imaging. This opens up the possibility for carrying out live axonal transport and synaptic imaging assays in future studies, in addition to the applications reported in this study. Graphical abstract Graphical abstract abbreviations: Channelrhodopsin-2 (CHR2+), pluripotent stem cell (PSC), motor neurons (MNs), myofibers (MFs), neuromuscular junction (NMJ)., Competing Interests: Competing interests* Y.-Y.L. is the principal investigator of a research grant received from Pfizer., (Copyright © 2023 The Authors; This is an open access article under the CC BY license ( https://creativecommons.org/licenses/by/4.0/).)

Blindness caused by advanced stages of inherited retinal diseases and age-related macular degeneration are characterized by photoreceptor loss. Cell therapy involving replacement with functional photoreceptor-like cells generated from human pluripotent stem cells holds great promise. Here, we generated a human recombinant retina-specific laminin isoform, LN523, and demonstrated the role in promoting the differentiation of human embryonic stem cells into photoreceptor progenitors. This chemically defined and xenogen-free method enables reproducible production of photoreceptor progenitors within 32 days. We observed that the transplantation into rd10 mice were able to protect the host photoreceptor outer nuclear layer (ONL) up to 2 weeks post transplantation as measured by full-field electroretinogram. At 4 weeks post transplantation, the engrafted cells were found to survive, mature, and associate with the host's rod bipolar cells. Visual behavioral assessment using the water maze swimming test demonstrated visual improvement in the cell-transplanted rodents. At 20 weeks post transplantation, the maturing engrafted cells were able to replace the loss of host ONL by extensive association with host bipolar cells and synapses. Post-transplanted rabbit model also provided congruent evidence for synaptic connectivity with the degenerated host retina. The results may pave the way for the development of stem cell-based therapeutics for retina degeneration., Competing Interests: Declaration of interests K.T. is co-founder and shareholder in BioLamina. K.T. and H.G.T. are co-founders of Alder Therapeutics AB. Other authors declare no competing interests., (Copyright © 2022 Duke-NUS Medical School. Published by Elsevier Inc. All rights reserved.)

Quantification of skeletal muscle functional contraction is essential to assess the outcomes of therapeutic procedures for neuromuscular disorders. Muscle three-dimensional "Organ-on-chip" models usually require a substantial amount of biological material, which rarely can be obtained from patient biopsies. Here, we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity at the single cell level. Optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled microenvironments, with myotubes derived from primary human myoblasts displaying spontaneous contractions. Analysis of nuclear morphology confirmed similar myonuclei structure between obtained myotubes and in vivo myofibers, as compared to 2D monolayers. LMNA-related Congenital Muscular Dystrophy (L-CMD) was modeled with successful development of diseased 3D myotubes displaying reduced contraction. The miniaturized myotube technology can thus be used to study contraction characteristics and evaluate how diseases affect muscle organization and force generation. Importantly, it requires significantly fewer starting materials than current systems, which should substantially improve drug screening capability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

Current imaging approaches limit the ability to perform multi-scale characterization of three-dimensional (3D) organotypic cultures (organoids) in large numbers. Here, we present an automated multi-scale 3D imaging platform synergizing high-density organoid cultures with rapid and live 3D single-objective light-sheet imaging. It is composed of disposable microfabricated organoid culture chips, termed JeWells, with embedded optical components and a laser beam-steering unit coupled to a commercial inverted microscope. It permits streamlining organoid culture and high-content 3D imaging on a single user-friendly instrument with minimal manipulations and a throughput of 300 organoids per hour. We demonstrate that the large number of 3D stacks that can be collected via our platform allows training deep learning-based algorithms to quantify morphogenetic organizations of organoids at multi-scales, ranging from the subcellular scale to the whole organoid level. We validated the versatility and robustness of our approach on intestine, hepatic, neuroectoderm organoids and oncospheres., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Adverse cutaneous reactions are potentially life-threatening skin side effects caused by drugs administered into the human body. The availability of a human-specific in vitro platform that can prospectively screen drugs and predict this risk is therefore of great importance to drug safety. However, since adverse cutaneous drug reactions are mediated by at least 2 distinct mechanisms, both involving systemic interactions between liver, immune and dermal tissues, existing in vitro skin models have not been able to comprehensively recapitulate these complex, multi-cellular interactions to predict the skin-sensitization potential of drugs. Here, we report a novel in vitro drug screening platform, which comprises a microfluidic multicellular coculture array (MCA) to model different mechanisms-of-action using a collection of simplistic cellular assays. The resultant readouts are then integrated with a machine-learning algorithm to predict the skin sensitizing potential of systemic drugs. The MCA consists of 4 cell culture compartments connected by diffusion microchannels to enable crosstalk between hepatocytes that generate drug metabolites, antigen-presenting cells (APCs) that detect the immunogenicity of the drug metabolites, and keratinocytes and dermal fibroblasts, which collectively determine drug metabolite-induced FasL-mediated apoptosis. A single drug screen using the MCA can simultaneously generate 5 readouts, which are integrated using support vector machine (SVM) and principal component analysis (PCA) to classify and visualize the drugs as skin sensitizers or non-skin sensitizers. The predictive performance of the MCA and SVM classification algorithm is then validated through a pilot screen of 11 drugs labelled by the US Food and Drug Administration (FDA), including 7 skin-sensitizing and 4 non-skin sensitizing drugs, using stratified 4-fold cross-validation (CV) on SVM. The predictive performance of our in vitro model achieves an average of 87.5% accuracy (correct prediction rate), 75% specificity (prediction rate of true negative drugs), and 100% sensitivity (prediction rate of true positive drugs). We then employ the MCA and the SVM training algorithm to prospectively identify the skin-sensitizing likelihood and mechanism-of-action for obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist which has undergone clinical trials for non-alcoholic steatohepatitis (NASH) with well-documented cutaneous side effects.

Hyperspectral microscopy is an imaging technique that provides spectroscopic information with high spatial resolution. When applied in the relevant wavelength region, such as in the infrared (IR), it can reveal a rich spectral fingerprint across different regions of a sample. Challenges associated with low efficiency and high cost of IR light sources and detector arrays have limited its broad adoption. Here, we introduce a new approach to IR hyperspectral microscopy, where the IR spectral map is obtained with off-the-shelf components built for visible light. The method is based on the nonlinear interference of correlated photons generated via parametric down-conversion. In this proof-of-concept we demonstrate the chemical mapping of a patterned sample, where different areas have distinctive IR spectroscopic fingerprints. The method provides a wide field of view, fast readout, and negligible heat delivered to the sample, which opens prospects for its further development for applications in material and biological studies., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Luminescent metal-organic frameworks (MOFs) are known to spontaneously self-assemble on human fingerprints. Here, we investigate the different chemical components of fingerprints and determine that MOF growth is predominantly induced by insoluble fatty acids. This finding shows that these simple biomolecules can be employed for the precise positioning of luminescent MOFs.

We demonstrate millimeters-long VCSEL linear arrays with SU-8 epoxy-based microlenses that are directly patterned and cross-linked on the output apertures by a simple, photoacid-diffusion-aided photolithography technique. The linear arrays are capable of delivering >7 W of peak pulsed output power. By exploiting the photoacid diffusion effect, it is possible to produce a range of microlens structures with height and radius of curvature ranging from approximately ten to tens of microns. Simulation and experimental results show that the far-field beam divergence can be reduced by a factor of up to 7 in VCSELs integrated with optimal microlens dimensions.