Efficient separation of large particles and giant cancer cells using an isosceles trapezoidal spiral microchannel.

Polyploid giant cancer cells (PGCCs) contribute to the genetic heterogeneity and evolutionary dynamics of tumors. Their size, however, complicates their isolation from mainstream tumor cell populations. Standard techniques like fluorescence-activated cell sorting (FACS) rely on fluorescent labeling, introducing potential challenges in subsequent PGCC analyses. In response, we developed the Isosceles Trapezoidal Spiral Microchannel (ITSµC), a microfluidic device optimizing the Dean drag force (FD) and exploiting uniform vortices for enhanced separation. Numerical simulations highlighted ITSµC's advantage in producing robust FD compared to rectangular and standard trapezoidal channels. Empirical results confirmed its ability to segregate larger polystyrene (PS) particles (avg. diameter: 50 µm) toward the inner wall, while directing smaller ones (avg. diameter: 23 µm) outward. Utilizing ITSµC, we efficiently isolated PGCCs from doxorubicin-resistant triple-negative breast cancer (DOXR-TNBC) and patient-derived cancer (PDC) cells, achieving outstanding purity, yield, and viability rates (all greater than 90%). This precision was accomplished without fluorescent markers, and the versatility of ITSµC suggests its potential in differentiating a wide range of heterogeneous cell populations.

Plant-Derived Anti-Human Epidermal Growth Factor Receptor 2 Antibody Suppresses Trastuzumab-Resistant Breast Cancer with Enhanced Nanoscale Binding.

Traditional monoclonal antibodies such as Trastuzumab encounter limitations when treating Human Epidermal Growth Factor Receptor 2 (HER2)-positive breast cancer, particularly in cases that develop resistance. This study introduces plant-derived anti-HER2 variable fragments of camelid heavy chain domain (VHH) fragment crystallizable region (Fc) KEDL(K) antibody as a potent alternative for overcoming these limitations. A variety of biophysical techniques, in vitro assays, and in vivo experiments uncover the antibody's nanoscale binding dynamics with transmembrane HER2 on living cells. Single-molecule force spectroscopy reveals the rapid formation of two robust bonds, exhibiting approximately 50 pN force resistance and bond lifetimes in the second range. The antibody demonstrates a specific affinity for HER2-positive breast cancer cells, including those that are Trastuzumab-resistant. Moreover, in immune-deficient mice, the plant-derived anti-HER2 VHH-FcK antibody exhibits superior antitumor activity, especially against tumors that are resistant to Trastuzumab. These findings underscore the plant-derived antibody's potential as an impactful immunotherapeutic strategy for treating Trastuzumab-resistant HER2-positive breast cancer.

Exploration of drug resistance mechanisms in triple negative breast cancer cells using a microfluidic device and patient tissues.

Chemoresistance is a major cause of treatment failure in many cancers. However, the life cycle of cancer cells as they respond to and survive environmental and therapeutic stress is understudied. In this study, we utilized a microfluidic device to induce the development of doxorubicin-resistant (DOXR) cells from triple negative breast cancer (TNBC) cells within 11 days by generating gradients of DOX and medium. In vivo chemoresistant xenograft models, an unbiased genome-wide transcriptome analysis, and a patient data/tissue analysis all showed that chemoresistance arose from failed epigenetic control of the nuclear protein-1 (NUPR1)/histone deacetylase 11 (HDAC11) axis, and high NUPR1 expression correlated with poor clinical outcomes. These results suggest that the chip can rapidly induce resistant cells that increase tumor heterogeneity and chemoresistance, highlighting the need for further studies on the epigenetic control of the NUPR1/HDAC11 axis in TNBC.

Three-Dimensional Hanging Drop Spheroid Plates for Easy Chimeric Antigen Receptor (CAR) T Cytotoxicity Assay.

Chimeric antigen receptor (CAR) T cell therapy shows a highly effective therapeutic effect on B-cell malignancies. The tumor microenvironment (TME) of solid tumors in vivo poses a great challenge to CAR T cell therapy due to its complexity. Recently, tumor spheroids have attracted much attention because of their ability to recapitulate TME. However, the use of tumor spheroids for the CAR T cytotoxicity assay involves the difficult task of separating unbound T cells and dead tumor cells from the spheroids. Therefore, we developed a three-dimensional hanging spheroid plate (3DHSP) that facilitates spheroid formation and separation of unbound and dead cells from spheroids during cytotoxicity assays. In this work, detailed steps have been described for fabrication and operation of the 3DHSP. This new 3DHSP device is a 96-well plate in which each well consists of a hanging dripper and a spheroid separation plate. A tumor spheroid forms in a droplet hanging in the dripper and is mixed with CAR T cells. The mixture in the droplet is deposited into the spheroid separation plate by pipetting, and unbound and dead CAR T and tumor cells are detached from the spheroid and moved to the waste well in the plate by tilting the 3DHSP at 20°. The size of the spheroid can be used as a readout for CAR T cell cytotoxicity assay, suggesting that the 3DHSP does not require cumbersome fluorescent staining.

A 3D hanging spheroid-filter plate for high-throughput drug testing and CAR T cell cytotoxicity assay.

Tumour spheroids are widely used in immune cell cytotoxicity assays and anticancer drug testing, providing a physiologically relevant model replicating the tumour microenvironment. However, co-culture of immune and tumour cells complicates quantification of immune cell killing efficiency. We present a novel 3D hanging spheroid-filter plate that efficiently facilitates spheroid formation and separates unbound/dead cells during cytotoxicity assays. Optical imaging directly measures the cytotoxic effects of anti-cancer drugs on tumour spheroids, eliminating the need for live/dead fluorescent staining. This approach enables cost-effective evaluation of T-cell cytotoxicity with specific chimeric antigen receptors (CARs), enhancing immune cell-based assays and drug testing in three-dimensional tumour models.

Phenotypic assay for cytotoxicity assessment of Balamuthia mandrillaris against human neurospheroids.

Introduction: The phenotypic screening of drugs against Balamuthia mandrillaris, a neuropathogenic amoeba, involves two simultaneous phases: an initial step to test amoebicidal activity followed by an assay for cytotoxicity to host cells. The emergence of three-dimensional (3D) cell cultures has provided a more physiologically relevant model than traditional 2D cell culture for studying the pathogenicity of B. mandrillaris. However, the measurement of ATP, a critical indicator of cell viability, is complicated by the overgrowth of B. mandrillaris in coculture with host cells during drug screening, making it challenging to differentiate between amoebicidal activity and drug toxicity to human cells. Methods: To address this limitation, we introduce a novel assay that utilizes three-dimensional hanging spheroid plates (3DHSPs) to evaluate both activities simultaneously on a single platform. Results and discussion: Our study showed that the incubation of neurospheroids with clinically isolated B. mandrillaris trophozoites resulted in a loss of neurospheroid integrity, while the ATP levels in the neurospheroids decreased over time, indicating decreased host cell viability. Conversely, ATP levels in isolated trophozoites increased, indicating active parasite metabolism. Our findings suggest that the 3DHSP-based assay can serve as an endpoint for the phenotypic screening of drugs against B. mandrillaris, providing a more efficient and accurate approach for evaluating both parasite cytotoxicity and viability.

Multi-Inlet Spheroid Generator for High-Throughput Combinatorial Drug Screening Based on the Tumor Microenvironment.

Tumor spheroids are powerful tools for drug screening and understanding tumor physiology. Among spheroid formation methods, the hanging drop method is considered most suitable for high-throughput screening (HTS) of anticancer drugs because it does not require surface treatment. However, it still needs to increase the liquid-holding capacity because hanging drops often fall due to the increased pressure caused by the addition of drugs, cells, etc. Here, we report a multi-inlet spheroid generator (MSG) enabling the stable addition of liquid-containing drugs or cells into a spheroid through its side inlet. The MSG was able to load additional solutions through the side inlet without increasing the force applied to the hanging drop. The volume of the additional liquid was easily controlled by varying the diameter of the side inlet. Furthermore, the sequences of the solution injections were manipulated using multiple side inlets. The feasibility of the MSG in clinical application was demonstrated by testing the efficacy of drugs in patient-derived cancer (PDC) cells and controlling the stromal cell ratio in the tumor microenvironment (TME) containing spheroids. Our results suggest that the MSG is a versatile platform for HTS of anticancer drugs and recapitulating the TME.

High-Throughput Screening of Anti-cancer Drugs Using a Microfluidic Spheroid Culture Device with a Concentration Gradient Generator.

Tumor spheroid models are widely used for drug screening as in vitro models of the tumor microenvironment. There are various ways in which tumor spheroid models can be prepared, including the self-assembly of cells using low-adherent plates, micro-patterned plates, or hanging-drop plates. Recently, drug high-throughput screening (HTS) approaches have incorporated the use of these culture systems. These HTS culture systems, however, require complicated equipment, such as robot arms, detectors, and software for handling solutions and data processing. Here, we describe protocols that allow tumor spheroids to be tested with different concentrations of a drug in a parallel fashion using a microfluidic device that generates a gradient of anti-cancer drugs. This microfluidic spheroid culture device with a concentration gradient generator (µFSCD-CGG) enables the formation of 50 tumor spheroids and the testing of drugs at five different concentrations. First, we provide a protocol for the fabrication of the µFSCD-CGG, which has both a culture array in which tumor cells are injected and aggregate to form spheroids and a concentration gradient generator for drug testing. Second, we provide a protocol for tumor spheroid formation and HTS of anti-cancer drugs using the device. Finally, we provide a protocol for assessing the response of tumor spheroids at different drug concentrations. To address the needs of the pharmaceutical industry, this protocol can be used for various cell types, including stem cells, and the number of tumor spheroids and drug concentration ranges that can be tested in the µFSCD-CGG can be increased. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Fabrication of a microfluidic spheroid culture device with a concentration gradient generator (µFSCD-CGG) Basic Protocol 2: Seeding cells and formation of spheroids in the µFSCD-CGG Basic Protocol 3: Drug treatment and assessment of cell viability in the µFSCD-CGG.

3D hanging spheroid plate for high-throughput CAR T cell cytotoxicity assay.

BACKGROUND: Most high-throughput screening (HTS) systems studying the cytotoxic effect of chimeric antigen receptor (CAR) T cells on tumor cells rely on two-dimensional cell culture that does not recapitulate the tumor microenvironment (TME). Tumor spheroids, however, can recapitulate the TME and have been used for cytotoxicity assays of CAR T cells. But a major obstacle to the use of tumor spheroids for cytotoxicity assays is the difficulty in separating unbound CAR T and dead tumor cells from spheroids. Here, we present a three-dimensional hanging spheroid plate (3DHSP), which facilitates the formation of spheroids and the separation of unbound and dead cells from spheroids during cytotoxicity assays. RESULTS: The 3DHSP is a 24-well plate, with each well composed of a hanging dripper, spheroid wells, and waste wells. In the dripper, a tumor spheroid was formed and mixed with CAR T cells. In the 3DHSP, droplets containing the spheroids were deposited into the spheroid separation well, where unbound and dead T and tumor cells were separated from the spheroid through a gap into the waste well by tilting the 3DHSP by more than 20°. Human epidermal growth factor receptor 2 (HER2)-positive tumor cells (BT474 and SKOV3) formed spheroids of approximately 300-350 µm in diameter after 2 days in the 3DHSP. The cytotoxic effects of T cells engineered to express CAR recognizing HER2 (HER2-CAR T cells) on these spheroids were directly measured by optical imaging, without the use of live/dead fluorescent staining of the cells. Our results suggest that the 3DHSP could be incorporated into a HTS system to screen for CARs that enable T cells to kill spheroids formed from a specific tumor type with high efficacy or for spheroids consisting of tumor types that can be killed efficiently by T cells bearing a specific CAR. CONCLUSIONS: The results suggest that the 3DHSP could be incorporated into a HTS system for the cytotoxic effects of CAR T cells on tumor spheroids.

Pillar-Based Mechanical Induction of an Aggressive Tumorigenic Lung Cancer Cell Model.

Tissue microarchitecture imposes physical constraints to the migration of individual cells. Especially in cancer metastasis, three-dimensional structural barriers within the extracellular matrix are known to affect the migratory behavior of cells, regulating the pathological state of the cells. Here, we employed a culture platform with micropillar arrays of 2 µm diameter and 16 µm pitch (2.16 micropillar) as a mechanical stimulant. Using this platform, we investigated how a long-term culture of A549 human lung carcinoma cells on the (2.16) micropillar-embossed dishes would influence the pathological state of the cell. A549 cells grown on the (2.16) micropillar array with 10 µm height exhibited a significantly elongated morphology and enhanced migration even after the detachment and reattachment, as evidenced in the conventional wound-healing assay, single-cell tracking analysis, and in vivo tumor colonization assays. Moreover, the pillar-induced morphological deformation in nuclei was accompanied by cell-cycle arrest in the S phase, leading to suppressed proliferation. While these marked traits of morphology-migration-proliferation support more aggressive characteristics of metastatic cancer cells, typical indices of epithelial-mesenchymal transition were not found, but instead, remarkable traces of amoeboidal transition were confirmed. Our study also emphasizes the importance of mechanical stimuli from the microenvironment during pathogenesis and how gained traits can be passed onto subsequent generations, ultimately affecting their pathophysiological behavior. Furthermore, this study highlights the potential use of pillar-based mechanical stimuli as an in vitro cell culture strategy to induce more aggressive tumorigenic cancer cell models.

A Golgi Apparatus-Targeting, Naphthalimide-Based Fluorescent Molecular Probe for the Selective Sensing of Formaldehyde.

Formaldehyde (FA) is a colorless, flammable, foul-smelling chemical used in building materials and in the production of numerous household chemical goods. Herein, a fluorescent chemosensor for FA is designed and prepared using a selective organ-targeting probe containing naphthalimide as a fluorophore and hydrazine as a FA-binding site. The amine group of the hydrazine reacts with FA to form a double bond and this condensation reaction is accompanied by a shift in the absorption band of the probe from 438 nm to 443 nm upon the addition of FA. Further, the addition of FA is shown to enhance the emission band at 532 nm relative to the very weak fluorescent emission of the probe itself. Moreover, a high specificity is demonstrated towards FA over other competing analytes such as the calcium ion (Ca2+), magnesium ion (Mg2+), acetaldehyde, benzaldehyde, salicylaldehyde, glucose, glutathione, sodium sulfide (Na2S), sodium hydrosulfide (NaHS), hydrogen peroxide (H2O2), and the tert-butylhydroperoxide radical. A typical two-photon dye incorporated into the probe provides intense fluorescence upon excitation at 800 nm, thus demonstrating potential application as a two-photon fluorescent probe for FA sensing. Furthermore, the probe is shown to exhibit a fast response time for the sensing of FA at room temperature and to facilitate intense fluorescence imaging of breast cancer cells upon exposure to FA, thus demonstrating its potential application for the monitoring of FA in living cells. Moreover, the presence of the phenylsulfonamide group allows the probe to visualize dynamic changes in the targeted Golgi apparatus. Hence, the as-designed probe is expected to open up new possibilities for unique interactions with organ-specific biological molecules with potential application in early cancer cell diagnosis.

Enhanced Luminescent Detection of Circulating Tumor Cells by a 3D Printed Immunomagnetic Concentrator.

Circulating tumor cells (CTCs) are an indicator of metastatic progression and relapse. Since non-CTC cells such as red blood cells outnumber CTCs in the blood, the separation and enrichment of CTCs is key to improving their detection sensitivity. The ATP luminescence assay can measure intracellular ATP to detect cells quickly but has not yet been used for CTC detection in the blood because extracellular ATP in the blood, derived from non-CTCs, interferes with the measurement. Herein, we report on the improvement of the ATP luminescence assay for the detection of CTCs by separating and concentrating CTCs in the blood using a 3D printed immunomagnetic concentrator (3DPIC). Because of its high-aspect-ratio structure and resistance to high flow rates, 3DPIC allows cancer cells in 10 mL to be concentrated 100 times within minutes. This enables the ATP luminescence assay to detect as low as 10 cells in blood, thereby being about 10 times more sensitive than when commercial kits are used for CTC concentration. This is the first time that the ATP luminescence assay was used for the detection of cancer cells in blood. These results demonstrate the feasibility of 3DPIC as a concentrator to improve the detection limit of the ATP luminescence assay for the detection of CTCs.

Probing mechanobiological role of filamin A in migration and invasion of human U87 glioblastoma cells using submicron soft pillars.

Filamin A (FLNa) belongs to an actin-binding protein family in binding and cross-linking actin filaments into a three-dimensional structure. However, little attention has been given to its mechanobiological role in cancer cells. Here, we quantitatively investigated the role of FLNa by analyzing the following parameters in negative control (NC) and FLNa-knockdown (KD) U87 glioma cells using submicron pillars (900 nm diameter and 2 µm height): traction force (TF), rigidity sensing ability, cell aspect ratio, migration speed, and invasiveness. During the initial phase of cell adhesion (< 1 h), FLNa-KD cells polarized more slowly than did NC cells, which can be explained by the loss of rigidity sensing in FLNa-KD cells. The higher motility of FLNa-KD cells relative to NC cells can be explained by the high TF exerted by FLNa-KD cells when compared to NC cells, while the higher invasiveness of FLNa-KD cells relative to NC cells can be explained by a greater number of filopodia in FLNa-KD cells than in NC cells. Our results suggest that FLNa plays important roles in suppressing motility and invasiveness of U87 cells.

Palladium Probe Consisting of Naphthalimide and Ethylenediamine for Selective Turn-On Sensing of CO and Cell Imaging.

An assay to detect carbon monoxide (CO), one of the gaseous signaling molecules, has been prepared using a new palladium complex probe. The ethylenediamine group linked to the naphthalimide fluorophore coordinates to Pd(II) which intramolecularly quenches the emission. Upon treatment with CO, the absorbance of the turn-on fluorescent sensor changes due to the formation of a complex between Pd(II) and CO at room temperature in a phosphate buffer. As the concentration of CO increases, the probe peak emission intensity at 527 nm gradually increases. Other analyte controls, such as K+, Mg2+, Al3+, Zn2+, Cr3+, Hg2+, Fe3+, alanine, glycine, leucine, lysine, serine, threonine, tyrosine, F-, Cl-, Br-, NO, NO2-, NO3-, HCO3-, CH3COO-, H2O2, •OH, and tBuOO•, exhibit no significant effect on emission intensity. The response time of the probe to CO was quite fast because of the relatively weak coordination of Pd(II) to the pendent ethylenediamine group. The Pd probe is capable of detecting CO in aqueous buffer as well as in living cells with high selectivity and stability, providing a potential real-time indicator for studying CO-involved reactions in biological systems.

Elucidating molecular mechanisms of acquired resistance to BRAF inhibitors in melanoma using a microfluidic device and deep sequencing.

BRAF inhibitors (e.g., vemurafenib) are widely used to treat metastatic melanoma with the BRAF V600E mutation. The initial response is often dramatic, but treatment resistance leads to disease progression in the majority of cases. Although secondary mutations in the mitogen-activated protein kinase signaling pathway are known to be responsible for this phenomenon, the molecular mechanisms governing acquired resistance are not known in more than half of patients. Here we report a genome- and transcriptome-wide study investigating the molecular mechanisms of acquired resistance to BRAF inhibitors. A microfluidic chip with a concentration gradient of vemurafenib was utilized to rapidly obtain therapy-resistant clones from two melanoma cell lines with the BRAF V600E mutation (A375 and SK-MEL-28). Exome and transcriptome data were produced from 13 resistant clones and analyzed to identify secondary mutations and gene expression changes. Various mechanisms, including phenotype switching and metabolic reprogramming, have been determined to contribute to resistance development differently for each clone. The roles of microphthalmia-associated transcription factor, the master transcription factor in melanocyte differentiation/dedifferentiation, were highlighted in terms of phenotype switching. Our study provides an omics-based comprehensive overview of the molecular mechanisms governing acquired resistance to BRAF inhibitor therapy.

Decrease in membrane fluidity and traction force induced by silica-coated magnetic nanoparticles.

BACKGROUND: Nanoparticles are being increasingly used in biomedical applications owing to their unique physical and chemical properties and small size. However, their biophysical assessment and evaluation of side-effects remain challenging. We addressed this issue by investigating the effects of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate [MNPs@SiO2(RITC)] on biophysical aspects, such as membrane fluidity and traction force of human embryonic kidney 293 (HEK293) cells. We further extended our understanding on the biophysical effects of nanoparticles on cells using a combination of metabolic profiling and transcriptomic network analysis. RESULTS: Overdose (1.0 µg/µL) treatment with MNPs@SiO2(RITC) induced lipid peroxidation and decreased membrane fluidity in HEK293 cells. In addition, HEK293 cells were morphologically shrunk, and their aspect ratio was significantly decreased. We found that each traction force (measured in micropillar) was increased, thereby increasing the total traction force in MNPs@SiO2(RITC)-treated HEK293 cells. Due to the reduction in membrane fluidity and elevation of traction force, the velocity of cell movement was also significantly decreased. Moreover, intracellular level of adenosine triphosphate (ATP) was also decreased in a dose-dependent manner upon treatment with MNPs@SiO2(RITC). To understand these biophysical changes in cells, we analysed the transcriptome and metabolic profiles and generated a metabotranscriptomics network, which revealed relationships among peroxidation of lipids, focal adhesion, cell movement, and related genes and metabolites. Furthermore, in silico prediction of the network showed increment in the peroxidation of lipids and suppression of focal adhesion and cell movement. CONCLUSION: Taken together, our results demonstrated that overdose of MNPs@SiO2(RITC) impairs cellular movement, followed by changes in the biophysical properties of cells, thus highlighting the need for biophysical assessment of nanoparticle-induced side-effects.

Fast and Easy Disinfection of Coronavirus-Contaminated Face Masks Using Ozone Gas Produced by a Dielectric Barrier Discharge Plasma Generator.

During the COVID-19 pandemic, face masks have become limited in stock. Most of sterilization methods are not applicable for eliminating virus from face masks without compromising the filtration efficiency of the masks. In this study, using a human coronavirus (HCoV-229E) as a surrogate for SARS-CoV-2 contamination on KF94 face masks, we show that the virus loses its infectivity with a 4 log reduction when exposed for 10 s to 120 ppm ozone gas produced by a dielectric barrier discharge plasma generator. Scanning electron microscopy, particulate filtration efficiency (PFE), and inhalation resistance tests revealed that there was no detectable structural or functional deterioration observed in the electrocharged filter layer of Korea Filter (KF) 94 masks even after their excessive exposure to ozone. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) showed decreases in amplification efficiency of HCoV-229E RNA recovered from masks exposed to ozone, indicating the damage to the RNA by the ozone treatment. Our results demonstrate that the plasma generator rapidly disinfects contaminated face masks at least five times without compromising filtration efficiency.

3D Bioprinted Vascularized Tumour for Drug Testing.

An in vitro screening system for anti-cancer drugs cannot exactly reflect the efficacy of drugs in vivo, without mimicking the tumour microenvironment (TME), which comprises cancer cells interacting with blood vessels and fibroblasts. Additionally, the tumour size should be controlled to obtain reliable and quantitative drug responses. Herein, we report a bioprinting method for recapitulating the TME with a controllable spheroid size. The TME was constructed by printing a blood vessel layer consisting of fibroblasts and endothelial cells in gelatine, alginate, and fibrinogen, followed by seeding multicellular tumour spheroids (MCTSs) of glioblastoma cells (U87 MG) onto the blood vessel layer. Under MCTSs, sprouts of blood vessels were generated and surrounding MCTSs thereby increasing the spheroid size. The combined treatment involving the anti-cancer drug temozolomide (TMZ) and the angiogenic inhibitor sunitinib was more effective than TMZ alone for MCTSs surrounded by blood vessels, which indicates the feasibility of the TME for in vitro testing of drug efficacy. These results suggest that the bioprinted vascularized tumour is highly useful for understanding tumour biology, as well as for in vitro drug testing.

Tauroursodeoxycholic acid attenuates cisplatin-induced hearing loss in rats.

Tauroursodeoxycholic acid (TUDCA) has been reported to be protective against apoptosis and oxidative stress in various cell types. A few studies have demonstrated otoprotective effects of TUDCA in mouse models. This study investigated the otoprotective effects of TUDCA in cisplatin (CXP)-induced hearing-loss rats. Eight-week-old female Sprague-Dawley rats were used. The CXP group received intraperitoneal injection of CXP at a dose of 5 mg/kg from day 1 to day 3. The CXP + TUDCA group received an intraperitoneal injection of 5 mg/kg CXP and 100 mg/kg TUDCA from day 1 to day 3. The mRNA expression levels of heme oxygenase 1 (HO1) and superoxide dismutase 2 (SOD2) were measured, and the protein levels of caspase 3, cleaved caspase 3, and aryl hydrocarbon receptor (AhR) were evaluated. The CXP group demonstrated higher mean auditory brainstem responses (ABR) thresholds than the control group. The mean ABR threshold shifts were lower in the CXP + TUDCA group than in the CXP group. The CXP group showed elevated HO1 and SOD2 mRNA expression levels compared to the control group, but these changes were reversed in the CXP + TUDCA group. Compared to the levels in the control group, caspase 3, cleaved caspase 3, and AhR levels were higher in the CXP group, but the increase in cleaved caspase-3 was attenuated in the CXP + TUDCA group. The cochlea showed a higher number of spiral ganglion cells and outer hair cells in the CXP + TUDCA group than in the CXP group. TUDCA reduced CXP-induced hearing loss in adult rats. The HO1-mediated antioxidative effects attenuated apoptosis in the cochlea, but AhR activation was not reversed.

Silica-Coated Magnetic Nanoparticles Decrease Human Bone Marrow-Derived Mesenchymal Stem Cell Migratory Activity by Reducing Membrane Fluidity and Impairing Focal Adhesion.

For stem cell-based therapies, the fate and distribution of stem cells should be traced using non-invasive or histological methods and a nanomaterial-based labelling agent. However, evaluation of the biophysical effects and related biological functions of nanomaterials in stem cells remains challenging. Here, we aimed to investigate the biophysical effects of nanomaterials on stem cells, including those on membrane fluidity, using total internal reflection fluorescence microscopy, and traction force, using micropillars of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) labelled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate (MNPs@SiO2(RITC)). Furthermore, to evaluate the biological functions related to these biophysical changes, we assessed the cell viability, reactive oxygen species (ROS) generation, intracellular cytoskeleton, and the migratory activity of MNPs@SiO2(RITC)-treated hBM-MSCs. Compared to that in the control, cell viability decreased by 10% and intracellular ROS increased by 2-fold due to the induction of 20% higher peroxidized lipid in hBM-MSCs treated with 1.0 µg/µL MNPs@SiO2(RITC). Membrane fluidity was reduced by MNPs@SiO2(RITC)-induced lipid oxidation in a concentration-dependent manner. In addition, cell shrinkage with abnormal formation of focal adhesions and ~30% decreased total traction force were observed in cells treated with 1.0 µg/µL MNPs@SiO2(RITC) without specific interaction between MNPs@SiO2(RITC) and cytoskeletal proteins. Furthermore, the migratory activity of hBM-MSCs, which was highly related to membrane fluidity and cytoskeletal abnormality, decreased significantly after MNPs@SiO2(RITC) treatment. These observations indicated that the migratory activity of hBM-MSCs was impaired by MNPs@SiO2(RITC) treatment due to changes in stem-cell biophysical properties and related biological functions, highlighting the important mechanisms via which nanoparticles impair migration of hBM-MSCs. Our findings indicate that nanoparticles used for stem cell trafficking or clinical applications should be labelled using optimal nanoparticle concentrations to preserve hBM-MSC migratory activity and ensure successful outcomes following stem cell localisation.