Satoshi Fujita

An aberrant wound-healing response to chronic liver injury results in excessive accumulation of extracellular matrix proteins, including collagen, leading to liver fibrosis, a process in which hepatocytes and hepatic stellate cells (HSCs) play key roles. In this study, we present HepG2 and LX-2 cocultured spheroids as a three-dimensional in vitro model of liver fibrosis and highlight the potential of hyperspectral Raman imaging as a label-free analytical approach for assessing fibrosis-associated responses. HepG2 cells and LX-2 cells served as hepatocytes and HSCs, respectively, within the coculture spheroids. Hyperspectral Raman imaging allowed for the acquisition of fingerprint spectra from individual cells, revealing distinct cytochrome-related band patterns that facilitated cell-type segmentation without labeling. To induce fibrosis, spheroids were treated with fibrosis activation factors: free fatty acids (FFAs) and transforming growth factor-β (TGF-β). FFA administration induced steatotic features in the coculture spheroids, as demonstrated by a direct correlation between the increasing lipid band intensity in the Raman spectra and FFA concentrations. Concurrently, a reduced cytochrome-related band intensity was observed in these spheroids. Furthermore, LX-2 spheroids but not HepG2 spheroids exhibited signs of fibrosis after TGF-β activation. Raman analysis revealed that the amide I and III bands reflected extracellular matrix, especially collagen accumulation, applied as fibrosis markers. Notably, in the 2:1 coculture spheroids, we observed enhanced fibrosis-related bands in the LX-2-enriched regions. The consistency between Raman imaging results and immunofluorescent images underscores the potential of hyperspectral Raman imaging as a sensitive and noninvasive method for assessing liver fibrosis.

Macrocyclic peptides have garnered increasing attention due to their therapeutic potential. However, the low cell membrane permeability of macrocyclic peptides limits their access to intracellular targets. This study aimed to apply live-cell Raman imaging to detect intracellular macrocyclic peptides as a potential therapeutic modality. Alkyne-tags with specific Raman bands were attached to several types of macrocyclic peptides originally designed as HIV protease inhibitors. Membrane permeability was measured by using the traditional penetration test. Variations in permeability were observed among the compounds, with the alkyne group showing a minimal effect on the results. Macrocyclic peptides with an alkyne-tag, which exhibited no permeability in the penetration test, were added to the live cells and evaluated using Raman imaging. Two-dimensional (2D) and three-dimensional (3D) Raman imaging detected the macrocyclic peptides in the intracellular region. However, the alkyne signal was not observed in cells treated with the macrocyclic peptides, showing cell penetration in the penetration test. These results are consistent with the observed pharmacological activities and suggest that the traditional penetration test may be insufficient for effective drug design of macrocyclic peptides. We conclude that combining emerging techniques, such as Raman imaging, with traditional methods can provide a more detailed evaluation of the permeability of macrocyclic peptides.

An ordered multispectral array of Au coated ellipse nanopillars was successfully fabricated via laser interference and nanoimprint lithography. The fabricated plasmonic substrate features a gradient plasmonic response in both translational and rotational directions across the chip, relative to a polarized UV–vis light source. Structural characterization of the substrate suggests that the gradient response is induced by the anisotropic valleys connecting the ellipse nanopillars in a hexagonal arrangement, resulting in varying nanopillar relative heights in various cross sections of the array. These gradual geometrical changes in the nanostructure allow control of the plasmonic response through two degrees of freedom (e.g., spatial translation or source polarization) on a single nanoimprinted chip. We also show that the fabricated substrate is viable for biosensing applications.

We present a facile method to fabricate stable Ca-alginate hydrogel domes covalently immobilized on glass plates. The system supports non-adherent cell proliferation and reagent delivery, offering a platform for high-throughput analysis.

Tissue-conserving partial mastectomy surgeries are often used to treat breast cancer, in which malignancies and surrounding margin tissues are removed. Morphologies of margin tissues are often assessed intraoperatively by a histopathologist, requiring excision and time-consuming preparation for microscopic examination. Changes in morphology often appear only after the mutation of cells involved in tumor propagation, suggesting that margins and perhaps conserved tissue may already contain cancer-converted cells prior to morphological changes. In a process known as field cancerization (FC), benign tissue surrounding a malignancy is affected by continued exposure to carcinogens, triggering cancer-promoting alterations like conversions of normal adipocytes and fibroblasts to cancer-associated adipocytes and fibroblasts. We report successful FC detection in human breast tissue samples excised during breast-conserving surgeries in human patients using Raman hyperspectral imaging and artificial neural networks (ANNs). Spectra in the images are segmented based on tissue modalities such as adipose, connective, and glandular tissues using intensities at Raman shifts of known molecules and label propagation, a method of transductive learning. ANNs that discriminate cancer from noncancer spectra return high quality (>95% accuracy) and reproducible distinction of spectra originating from cancerous tissue at high signal-to-error ratios (S/Es). Good distinction is returned in all modalities, with adipose-rich modalities returning the most reliable classifications, suggesting reliable discrimination of normal and cancer-associated adipocytes. Spectral S/Es are increased using larger spectral measurement areas, but this causes spectral mixing across different tissue modalities and significant biological variation, failing to deliver a corresponding increase in classification quality.

DNA microarrays have been applied for comprehensive genotyping, but remain a drawback in complicated operations. As a solution, we previously reported the solid-phase collateral cleavage (SPCC) system based on the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 12 (CRISPR/Cas12). Surface-immobilized Cas12-CRISPR RNA (crRNA) can directly hybridize target double-stranded DNA (dsDNA) and subsequently produce a signal via the cleavage of single-stranded DNA (ssDNA) reporter immobilized on the same spot. Therefore, SPCC-based multiplex dsDNA detection can be performed easily. This study reports the miniaturization of SPCC-based spots patterned by a non-contact printer and its performance in comprehensive genotyping on a massively accumulated array. Initially, printing, immobilization, and washing processes of Cas12–crRNA were established to fabricate the non-contact-patterned SPCC-based sensor array. A target dsDNA concentration response was obtained based on the developed sensor array, even with a spot diameter of 0.64 ± 0.05 mm. Also, the limit of detection was 572 pM, 531 pM, and 3.04 nM with 40, 20, and 10 nL-printing of Cas12–crRNA, respectively. Furthermore, the sensor array specifically detected three dsDNA sequences in one-pot multiplexing; therefore, the feasibility of comprehensive genotyping was confirmed. These results demonstrate that our technology can be miniaturized as a CRISPR/Cas12-based microarray by using non-contact printing. In the future, the non-contact-patterned SPCC-based sensor array can be applied as an alternative tool to DNA microarrays.

The blood–brain barrier (BBB) is a selective barrier that controls the transport between the blood and neural tissue features and maintains brain homeostasis to protect the central nervous system (CNS). In vitro models can be useful to understand the role of the BBB in disease and assess the effects of drug delivery. Recently, we reported a 3D BBB model with perfusable microvasculature in a Transwell insert. It replicates several key features of the native BBB, as it showed size-selective permeability of different molecular weights of dextran, activity of the P-glycoprotein efflux pump, and functionality of receptor-mediated transcytosis (RMT), which is the most investigated pathway for the transportation of macromolecules through endothelial cells of the BBB. For quality control and permeability evaluation in commercial use, visualization and quantification of the 3D vascular lumen structures is absolutely crucial. Here, for the first time, we report a rapid, non-invasive optical coherence tomography (OCT)-based approach to quantify the microvessel network in the 3D in vitro BBB model. Briefly, we successfully obtained the 3D OCT images of the BBB model and further processed the images using three strategies: morphological imaging processing (MIP), random forest machine learning using the Trainable Weka Segmentation plugin (RF-TWS), and deep learning using pix2pix cGAN. The performance of these methods was evaluated by comparing their output images with manually selected ground truth images. It suggested that deep learning performed well on object identification of OCT images and its computation results of vessel counts and surface areas were close to the ground truth results. This study not only facilitates the permeability evaluation of the BBB model but also offers a rapid, non-invasive observational and quantitative approach for the increasing number of other 3D in vitro models.

Human-hepatoblastoma-derived cell line, HepG2, has been widely used in liver and liver cancer studies. HepG2 spheroids produced in a three-dimensional (3D) culture system provide a better biological model than cells cultured in a two-dimensional (2D) culture system. Since cells at the center of spheroids exhibit specific behaviors attributed to hypoxic conditions, a 3D cell culture system that allows the observation of such cells using conventional optical or fluorescence microscopes would be useful. In this study, HepG2 cells were cultured in “Cell Dome”, a micro-dome in which cells are enclosed in a cavity consisting of a hemispherical hydrogel shell. HepG2 cells formed hemispherical cell aggregates which filled the cavity of Cell Domes on 18 days of culture and the cells could continue to be cultured for 29 days. The cells at the center of hemispherical cell aggregates were observed using a fluorescence microscope. The cells grew in Cell Domes for 18 days exhibited higher Pi-class Glutathione S-Transferase enzymatic activity, hypoxia inducible factor-1α gene expression, and higher tolerance to mitomycin C than those cultured in 2D on tissue culture dishes (* p < 0.05). These results indicate that the center of the glass adhesive surface of hemispherical cell aggregates which is expected to have the similar environment as the center of the spheroids can be directly observed through glass plates. In conclusion, Cell Dome would be useful as an evaluation platform for organized HepG2 cells.

Non-adherent cells, such as hematopoietic cells and lymphocytes, are important research subjects in medical and biological fields. Therefore, a system that enables the handling of non-adherent cells in solutions in the same manner as that of adhering cells during medium exchange, exposure to chemicals, washing, and staining in imaging applications would be useful. Here, we report a ‘Cell Dome’ platform in which non-adherent cells can be enclosed and grown in the cavities of about 1 mm diameter and 270 μm height. The domes consist of an alginate-based hydrogel shell of 90 μm thickness. Cell Domes were formed on glass plates by horseradish peroxidase-mediated cross-linking. Human leukaemia cell line K562 cells enclosed in Cell Domes were stable for 29 days with every 2–3 days of medium change. The enclosed cells grew in the cavities and were stained and differentiated with reagents supplied from the surrounding medium. Additionally, K562 cells that filled the cavities (a 3D microenvironment) were more hypoxic and highly resistant to mitomycin C than those cultured in 2D. These findings demonstrate that the ‘Cell Dome’ may be a promising tool for conveniently culturing and evaluating non-adherent cells.