Hybrid Raman and Laser-Induced Breakdown Spectroscopy for Food Authentication Applications.

The issue of food fraud has become a significant global concern as it affects both the quality and safety of food products, ultimately resulting in the loss of customer trust and brand loyalty. To address this problem, we have developed an innovative approach that can tackle various types of food fraud, including adulteration, substitution, and dilution. Our methodology utilizes an integrated system that combines laser-induced breakdown spectroscopy (LIBS) and Raman spectroscopy. Although both techniques emerged as valuable tools for food analysis, they have until now been used separately, and their combined potential in food fraud has not been thoroughly tested. The aim of our study was to demonstrate the potential benefits of integrating Raman and LIBS modalities in a portable system for improved product classification and subsequent authentication. In pursuit of this objective, we designed and tested a compact, hybrid Raman/LIBS system, which exhibited distinct advantages over the individual modalities. Our findings illustrate that the combination of these two modalities can achieve higher accuracy in product classification, leading to more effective and reliable product authentication. Overall, our research highlights the potential of hybrid systems for practical applications in a variety of industries. The integration and design were mainly focused on the detection and characterization of both elemental and molecular elements in various food products. Two different sets of solid food samples (sixteen Alpine-style cheeses and seven brands of Arabica coffee beans) were chosen for the authentication analysis. Class detection and classification were accomplished through the use of multivariate feature selection and machine-learning procedures. The accuracy of classification was observed to improve by approximately 10% when utilizing the hybrid Raman/LIBS spectra, as opposed to the analysis of spectra from the individual methods. This clearly demonstrates that the hybrid system can significantly improve food authentication accuracy while maintaining the portability of the combined system. Thus, the successful implementation of a hybrid Raman-LIBS technique is expected to contribute to the development of novel portable devices for food authentication in food as well as other various industries.

A single-cell surgery microfluidic device for transplanting tumor cytoplasm into dendritic cells without nuclei mixing.

This study aimed to demonstrate the feasibility of generating tumor cell vaccine models by single-cell surgery in a microfluidic device that integrates one-to-one electrofusion, shear flow reseparation, and on-device culture. The device was microfabricated from polydimethylsiloxane (PDMS) and consisted of microorifices (aperture size: ∼3 µm) for one-to-one fusion, and microcages for on-device culture. Using the device, we could achieve one-to-one electrofusion of leukemic plasmacytoid dendritic cells (DC-like cells) and Jurkat cells with a fusion efficiency of ∼ 80%. Fusion via the narrow microorifices allowed DC-like cells to acquire cytoplasmic contents of the Jurkat cells while preventing nuclei mixing. After fusion, the DC-like cells were selectively reseparated from the Jurkat cells by shear flow application to generate tumor nuclei-free antigen-recipient DC-like (tarDC-like) cells. When cultured as single cells on the device, these cells could survive under gentle medium perfusion with a median survival time of 11.5 h, although a few cells could survive longer than 36 h. Overall, this study demonstrates single-cell surgery in a microfluidic device for potential generation of dendritic cell vaccines which are uncontaminated with tumor nucleic materials. We believe that this study will inspire the generation of safer tumor cell vaccines for cancer immunotherapy.

Study of transmembrane ion transport under tonicity imbalance using a combination of low frequency-electrical impedance spectroscopy (LF-EIS) and improved ion transport model.

Transmembrane ion transport under tonicity imbalance has been investigated using a combination of low frequency-electrical impedance spectroscopy (LF-EIS) and improved ion transport model, by considering the cell diameterd[m] and the initial intracellular ion concentrationcin[mM] as a function of tonicity expressed by sucrose concentrationcs[mM]. The transmembrane ion transport is influenced by extracellular tonicity conditions, leading to a facilitation/inhibition of ion passage through the cell membrane. The transmembrane transport coefficientP[m s-1], which represents the ability of transmembrane ion transport, is calculated by the extracellular ion concentrations obtained by improved ion transport model and LF-EIS measurement.Pis calculated as 4.11 × 10-6and 3.44 × 10-6m s-1atcsof 10 and 30 mM representing hypotonic condition, 2.44 × 10-6m s-1atcsof 50 mM representing isotonic condition, and 3.68 × 10-6, 5.16 × 10-6, 9.51 × 10-6, and 14.89 × 10-6m s-1atcsof 75, 100, 125 and 150 mM representing hypertonic condition. The LF-EIS results indicate that the transmembrane ion transport is promoted under hypertonic and hypotonic conditions compared to isotonic condition. To verify the LF-EIS results, fluorescence intensityF[-] of extracellular potassium ions is observed to obtain the temporal distribution of average potassium ion concentration within the region of 3.6µm from cell membrane interfacecROI[mM]. The slopes of ∆cROI/cROI1to timetare 0.0003, 0.0002, and 0.0006 under hypotonic, isotonic, and hypertonic conditions, wherecROI1denotes initialcROI, which shows the same tendency with LF-EIS result that is verified by the potassium ion fluorescence observation.

Pluripotency state of mouse ES cells determines their contribution to self-organized layer formation by mesh closure on microstructured adhesion-limiting substrates.

Assembly of pluripotent stem cells to initiate self-organized tissue formation on engineered scaffolds is an important process in stem cell engineering. Pluripotent stem cells are known to exist in diverse pluripotency states, with heterogeneous subpopulations exhibiting differential gene expression levels, but how such diverse pluripotency states orchestrate tissue formation is still an unrevealed question. In this study, using microstructured adhesion-limiting substrates, we aimed to clarify the contribution to self-organized layer formation by mouse embryonic stem cells in different pluripotency states: ground and naïve state. We found that while ground state cells as well as sorted REX1-high expression cells formed discontinuous cell layers with limited lateral spread, naïve state cells could successfully self-organize to form a continuous layer by progressive mesh closure within 3 days. Using sequential immunofluorescence microscopy to examine the mesh closure process, we found that KRT8+ cells were particularly localized around unfilled holes, occasionally bridging the holes in a manner suggestive of their role in the closure process. These results highlight that compared with ground state cells, naïve state cells possess a higher capability to contribute to self-organized layer formation by mesh closure. Thus, this study provides insights with implications for the application of stem cells in scaffold-based tissue engineering.

Edge-localized alteration in pluripotency state of mouse ES cells forming topography-confined layers on designed mesh substrates.

Self-organization of pluripotent stem cells during tissue formation is directed by the adhesion microenvironment, which defines the resulting tissue topography. Although the influence of tissue topography on pluripotency state has been inferred, this aspect of self-organization remains largely unexplored. In this study, to determine the effect of self-organized tissue topography on pluripotency loss, we designed novel island mesh substrates to confine the self-organization process of mouse embryonic stem cells, enabling us to generate isolated cell layers with an island-like topography and overhanging edges. Using immunofluorescence microscopy, we determined that cells at the tissue edge exhibited deformed nuclei associated with low OCT3/4, in contrast with cells nested in the tissue interior which had round-shaped nuclei and exhibited sustained OCT3/4 expression. Interestingly, F-actin and phospho-myosin light chain were visibly enriched at the tissue edge where ERK activation and elevated AP-2γ expression were also found to be localized, as determined using both immunofluorescence microscopy and RT-qPCR analysis. Since actomyosin contractility is known to cause ERK activation, these results suggest that mechanical condition at the tissue edge can contribute to loss of pluripotency leading to differentiation. Thus, our study draws attention to the influence of self-organized tissue topography in stem cell culture and differentiation.

Nuclear transplantation between allogeneic cells through topological reconnection of plasma membrane in a microfluidic system.

Previous studies have demonstrated that somatic cells fused with pluripotent stem cells can be reprogrammed on the basis of reprogramming factors acquired from the latter. However, fusion-reprogrammed cells are deemed unsuitable for therapeutic applications mainly because conventional fusion techniques often yield tetraploid fusants that contain exogenous genes acquired from the fusion partners. Here, we present a novel cell-cell topological reconnection technique and demonstrate its application to nuclear transplantation between a somatic cell and a stem cell without nuclei mixing. As a proof of concept, a microfluidic fusion chip embodied with a microslit (4 µm in width) to prevent nuclei mixing was developed and used to perform one-to-one electrofusion of a target somatic cell (Jurkat cell) with an induced pluripotent stem (iPS) cell. To extract its cytoplasm, the target cell was first topologically connected to a sacrificial iPS cell by electrofusion via a microslit, followed by shear flow removal of the latter to obtain a cytoplasm-depleted nucleus of the target cell. Then, to replace the lost cytoplasm, topological reconnection to a second iPS cell was performed similarly by electrofusion, followed by shear flow separation of the target cell to enable it acquire most of the iPS cytoplasm, but without nuclei mixing. Microscopic observation of target cells harvested and cultured post hoc in a microwell confirmed that they manifested cell division. Taken together, these results demonstrate the potential application of the cell-cell topological reconnection technique to somatic cell nuclear transplantation for the generation of autologous pluripotent stem cells.

Modulation of adhesion microenvironment using mesh substrates triggers self-organization and primordial germ cell-like differentiation in mouse ES cells.

The cell adhesion microenvironment plays contributory roles in the induction of self-organized tissue formation and differentiation of pluripotent stem cells (PSCs). However, physical factors emanating from the adhesion microenvironment have been less investigated largely in part due to overreliance on biochemical approaches utilizing cytokines to drive in vitro developmental processes. Here, we report that a mesh culture technique can potentially induce mouse embryonic stem cells (mESCs) to self-organize and differentiate into cells expressing key signatures of primordial germ cells (PGCs) even with pluripotency maintained in the culture medium. Intriguingly, mESCs cultured on mesh substrates consisting of thin (5 µm-wide) strands and considerably large (200 µm-wide) openings which were set suspended in order to minimize the cell-substrate adhesion area, self-organized into cell sheets relying solely on cell-cell interactions to fill the large mesh openings by Day 2, and further into dome-shaped features around Day 6. Characterization using microarray analysis and immunofluorescence microscopy revealed that sheet-forming cells exhibited differential gene expressions related to PGCs as early as Day 2, but not other lineages such as epiblast, primitive endoderm, and trophectoderm, implying that the initial interaction with the mesh microenvironment and subsequent self-organization into cells sheets might have triggered PGC-like differentiation to occur differently from the previously reported pathway via epiblast-like differentiation. Overall, considering that the observed differentiation occurred without addition of known biochemical inducers, this study highlights that bioengineering techniques for modulating the adhesion microenvironment alone can be harnessed to coax PSCs to self-organize and differentiate, in this case, to a PGC-like state.

A microfluidic device for isolating intact chromosomes from single mammalian cells and probing their folding stability by controlling solution conditions.

Chromatin folding shows spatio-temporal fluctuations in living undifferentiated cells, but fixed spatial heterogeneity in differentiated cells. However, little is known about variation in folding stability along the chromatin fibres during differentiation. In addition, effective methods to investigate folding stability at the single cell level are lacking. In the present study, we developed a microfluidic device that enables non-destructive isolation of chromosomes from single mammalian cells as well as real-time microscopic monitoring of the partial unfolding and stretching of individual chromosomes with increasing salt concentrations under a gentle flow. Using this device, we compared the folding stability of chromosomes between non-differentiated and differentiated cells and found that the salt concentration which induces the chromosome unfolding was lower (≤500 mM NaCl) for chromosomes derived from undifferentiated cells, suggesting that the chromatin folding stability of these cells is lower than that of differentiated cells. In addition, individual unfolded chromosomes, i.e., chromatin fibres, were stretched to 150-800 µm non-destructively under 750 mM NaCl and showed distributions of highly/less folded regions along the fibres. Thus, our technique can provide insights into the aspects of chromatin folding that influence the epigenetic control of cell differentiation.

Self-organization of human iPS cells into trophectoderm mimicking cysts induced by adhesion restriction using microstructured mesh scaffolds.

Cellular dynamics leading to the formation of the trophectoderm in humans remain poorly understood owing to limited accessibility to human embryos for research into early human embryogenesis. Compared to animal models, organoids formed by self-organization of stem cells in vitro may provide better insights into differentiation and complex morphogenetic processes occurring during early human embryogenesis. Here we demonstrate that modulating the cell culture microenvironment alone can trigger self-organization of human induced pluripotent stem cells (hiPSCs) to yield trophectoderm-mimicking cysts without chemical induction. To modulate the adhesion microenvironment, we used the mesh culture technique recently developed by our group, which involves culturing hiPSCs on suspended micro-structured meshes with limited surface area for cell adhesion. We show that this adhesion-restriction strategy can trigger a two-stage self-organization of hiPSCs; first into stem cell sheets, which express pluripotency signatures until around day 8-10, then into spherical cysts following differentiation and self-organization of the sheet-forming cells. Detailed morphological analysis using immunofluorescence microscopy with both confocal and two-photon microscopes revealed the anatomy of the cysts as consisting of a squamous epithelial wall richly expressing E-cadherin and CDX2. We also confirmed that the cysts exhibit a polarized morphology with basal protrusions, which show migratory behavior when anchored. Together, our results point to the formation of cysts which morphologically resemble the trophectoderm at the late-stage blastocyst. Thus, the mesh culture microenvironment can initiate self-organization of hiPSCs into trophectoderm-mimicking cysts as organoids with potential application in the study of early embryogenesis and also in drug development.

Nucleosomes Exhibit Non-uniform Unwrapping Along Native Chromatin Fibers with Increasing Salt Concentration as Revealed by Direct Imaging in a Microfluidic Channel.

Identifying the distribution of the higher-order structure of chromatin - a complex of DNA and proteins - along genomic DNA can clarify the mechanisms underlying cell development and differentiation, including gene regulation. However, genome-wide analysis of this distribution at the single-cell level remains an outstanding challenge. Here, the authors report a new method for investigating changes in and the distribution of higher-order structures along native chromatin fibers - ranging over 100 µm in length - relative to changes in salt concentration. To this end, the authors developed a microfluidic platform that enabled us to isolate chromatin fibers from single cells and tether them to microstructures in a microfluidic channel without fragmentation. The fibers were then exposed to varying concentrations of salt solution under microscopic observation. As a result, the fibers are non-uniformly elongated by up to 2-3 times along the fiber axis as salt concentration was increased from 0 to 3 M, suggesting that chromosome structural stability is non-uniformly distributed along chromatin fibers in their native form. Thus, our system enables direct microscopic analysis of individual chromatin fibers from single cells, which can provide insights into epigenetic mechanisms of cell development, cell differentiation, and carcinogenesis.

Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells.

Mechanical methods for inducing differentiation and directing lineage specification will be instrumental in the application of pluripotent stem cells. Here, we demonstrate that minimization of cell-substrate adhesion can initiate and direct the differentiation of human pluripotent stem cells (hiPSCs) into cyst-forming trophoblast lineage cells (TLCs) without stimulation with cytokines or small molecules. To precisely control cell-substrate adhesion area, we developed a novel culture method where cells are cultured on microstructured mesh sheets suspended in a culture medium such that cells on mesh are completely out of contact with the culture dish. We used microfabricated mesh sheets that consisted of open meshes (100∼200 µm in pitch) with narrow mesh strands (3-5 µm in width) to provide support for initial cell attachment and growth. We demonstrate that minimization of cell adhesion area achieved by this culture method can trigger a sequence of morphogenetic transformations that begin with individual hiPSCs attached on the mesh strands proliferating to form cell sheets by self-assembly organization and ultimately differentiating after 10-15 days of mesh culture to generate spherical cysts that secreted human chorionic gonadotropin (hCG) hormone and expressed caudal-related homeobox 2 factor (CDX2), a specific marker of trophoblast lineage. Thus, this study demonstrates a simple and direct mechanical approach to induce trophoblast differentiation and generate cysts for application in the study of early human embryogenesis and drug development and screening.

Dynamic coupling between actin network flow and turnover revealed by flow mapping in the lamella of crawling fragments.

Dynamic turnover and transport of actin filament network is essential for protrusive force generation and traction force development during cell migration. To elucidate the dynamic coupling between actin network flow and turnover, we focused on flow dynamics in the lamella of one of the simplest but elegant motility systems; crawling fragments derived from fish keratocytes. Interestingly, we show that actin network in the lamella of fragments is not stationary as earlier reported, but exhibits a flow dynamics that is strikingly similar to that reported for higher order cells, suggesting that network flow is an intrinsic property of the actin cytoskeleton that is fundamental to cell migration. We also demonstrate that whereas polymerization mediates network assembly at the front, surprisingly, network flow convergence modulates network disassembly toward the rear of the lamella, suggesting that flow and turnover are coupled during migration. These results obtained using simple motility systems are significant to the understanding of actin network dynamics in migrating cells, and they will be found useful for developing biophysical models for elucidating the fundamental mechanisms of cell migration.

Actomyosin contractility spatiotemporally regulates actin network dynamics in migrating cells.

Coupling interactions among mechanical and biochemical factors are important for the realization of various cellular processes that determine cell migration. Although F-actin network dynamics has been the focus of many studies, it is not yet clear how mechanical forces generated by actomyosin contractility spatiotemporally regulate this fundamental aspect of cell migration. In this study, using a combination of fluorescent speckle microscopy and particle imaging velocimetry techniques, we perturbed the actomyosin system and examined quantitatively the consequence of actomyosin contractility on F-actin network flow and deformation in the lamellipodia of actively migrating fish keratocytes. F-actin flow fields were characterized by retrograde flow at the front and anterograde flow at the back of the lamellipodia, and the two flows merged to form a convergence zone of reduced flow intensity. Interestingly, activating or inhibiting actomyosin contractility altered network flow intensity and convergence, suggesting that network dynamics is directly regulated by actomyosin contractility. Moreover, quantitative analysis of F-actin network deformation revealed that the deformation was significantly negative and predominant in the direction of cell migration. Furthermore, perturbation experiments revealed that the deformation was a function of actomyosin contractility. Based on these results, we suggest that the actin cytoskeletal structure is a mechanically self-regulating system, and we propose an elaborate pathway for the spatiotemporal self-regulation of the actin cytoskeletal structure during cell migration. In the proposed pathway, mechanical forces generated by actomyosin interactions are considered central to the realization of the various mechanochemical processes that determine cell motility.

Strain field in actin filament network in lamellipodia of migrating cells: implication for network reorganization.

Cell motility is spatiotemporally regulated by interactions among mechanical and biochemical factors involved in the regulation of cytoskeletal actin structure reorganization. Although the molecular mechanisms underlying cell motility have been well investigated, the contributions of mechanical factors such as strain in the network reorganization remain unclear. In this study, we have quantitatively evaluated the strain field in the actin filament network forming the lamellipodia of migrating fish keratocytes to elucidate the mechanism by which actin filament network reorganization is regulated by biomechanical factors. The results highlight the existence of a negative (compressive) strain in the lamellipodia whose direction is parallel to that of cell movement. A close correlation was found between the distributions of the strain and the actin filament density in the lamellipodia, suggesting that negative strain may be involved in filament depolymerization. Based on this result, we propose a selective depolymerization model which suggests that negative strain may couple with biomechanical factors such as ADF/cofilin to promote selective depolymerization of filaments oriented in the direction of the deformation because such filaments experience relatively higher levels of the deformation. This model, in conjunction with others, may explain the observed reduction in filament density and the reorganization of actin filament network at the back of the lamellipodia of migrating fish keratocytes. Thus, we suggest that by coupling with biochemical factors, mechanical factors are involved in the regulation of actin filament depolymerization, thereby contributing to the regulation of cell motility.