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Abstract Background We developed a Rare Cell Sorter (RCS) for collecting single cell including circulating tumor cells (CTCs). This single-institution pilot study evaluated the ability of this device to detect tumor-like cells in patients with lung cancer and confirmed their genuineness based on the epidermal growth factor receptor (EGFR) mutation concordance with tissue samples. Methods This study included patients treated for lung cancer from September 2021 to August 2022 in University of Tsukuba Hospital. Peripheral blood samples were obtained before surgery or during periodic medical checks for patients treated with drugs. We used the RCS to capture cells based on size. The cells were stained, and the Hoechst-positive, CD45-negative, and epithelial celladhesion molecule (EpCAM)- positive cells were defined as CTCs, were collected. The presumptive CTCs were counted and tested using digital droplet polymerase chain reaction for EGFR mutations and compared with the tissue EGFR status to check concordance. Results Eighteen patients were included in this study and CTCs were detected in 6 patients (33%). The CTCs from three patients showed EGFR mutation, and the EGFR mutation status of CTCs concorded with that of tissue samples in 83% of the cases (5/6). Only one CTC showed a different status from the tissue, and the concordance rate of EGFR status between CTCs and the tissue was 96% (24/25). Conclusion The ability of the RCS to detect CTCs in patients with lung cancer was demonstrated based on the concordance of EGFR status in this pilot study. This novel hybrid method of CTC recovery using the RCS has the potential to recover a wide range of CTCs regardless of EpCAM. Further validation through a large-scale study is needed.

Abstract The mechanical properties of cells, such as leukocytes, in a diseased state differ from those of healthy cells, typically due to their microstructure. The deformability of the cells through a constrictive area is analyzed by the applied stress to the cell. This study investigates the relationship between the sample flow speed and distribution of captured leukocytes based on the cell deformability using a microfluidic device. The device comprises of microfilters that serve as the filtration mechanism. The microfilter gap size gradually decreases from 15 to 3 µm to facilitate the deformability-based separation. Leukocytes have various sizes; hence, they can be separated by microfilters directly from whole blood samples without any cell clogging, and they do not require sample pre-processing such as centrifugation or red blood cell lysis. The distribution of leukocytes captured by the microfilters with respect to the sample flow speed can be analyzed; at higher sample flow speeds of 6 µL/min, small leukocytes with a size of 7 µm could not be captured and they passed through the smallest microfilter gap size of 3 µm. For smaller leukocytes, such as lymphocytes, the distributions are mainly at gap sizes of 4 µm to 8 µm, with most of the lymphocytes captured at the 6 µm microfilter gap size. We conclude that the distribution of the cells captured during the filtration varies depending on the microfilter gap sizes, applied sample flow speed, cell sizes, and the ability of the cells to deform. The deformability imaging profiles of the sample could be developed from the images of the cell distribution, which might be useful for preliminary screening in the clinical applications. This work presents the development of a simple device for the study of cell deformability as the results provide a biophysical marker in high throughput and bulk sample analyses.

Abstract Providing an accurate count of total leukocytes and specific subsets (such as T-cells and B-cells) within small amounts of whole blood is a rather challenging ordeal due to the lack of techniques that enable the separation of leukocytes from a limited amount of whole blood. In a previous study we designed a microfluidic chip utilizing a micropillar array to isolate T-cells and B-cells from the sub-microliter of whole blood. Due to the variability of cells in size, morphology and color intensity, a Histogram of Oriented Gradients (HOG) based Support Vector Machine (SVM) classifier was proposed with an average accuracy of 94%, specificity of 99% and sensitivity of 90%. The HOG can separate the cells from the background with a high accuracy rate however, some noise is similar in shape and size to the actual cells and this results in misclassification. To alleviate this situation, in this study a convolutional neural network is trained and used to distinguish T-cells and B-cells with an accuracy rate of 98%, a specificity of 99% and a sensitivity of 97%. We also propose an HOG feature based SVM classifier to preselect the detection windows to accelerate the detection to process images in less than 10 min. The proposed on-chip cell detecting and counting method will be useful for numerous applications in diagnosis and for monitoring diseases.

Abstract Absolute counting of total leukocytes and specific subset (such as T-cells and B-cells) within small amounts of whole blood is difficult due to the lack of techniques that enables separation of leukocytes from limited volume of whole blood. In this study, a microfluidic chip equipped with a size controlled micropillar array for highly separation of T-cells and B-cells from sub-microliter of whole blood was studied. Based on the difference in size and deformability, leukocytes were separated from other blood cells by micropillar arrays. However, the variability of cells in size, morphology and color intensity along with the spectrum crosstalk between fluorescence dyes make cell detection among pillars extremely difficult. In this paper, an support vector machine supervised machine learning classifier based on both Histogram of Oriented Gradients (HOG) and color distribution features was proposed to distinguish T-cells and B-cells fast and robustly. HOG features were utilized to detect cells from background and noise; color distribution features were employed to alleviate the effect of fluorescence spectrum crosstalk. Experiment showed we achieved average detection accuracy of 94% for detecting T-cells and B-cells from the background. Furthermore, we also got 96% accuracy with cross validation to detect T-cells from B-cells. Both theoretical analysis and experiments demonstrated the proposed method and system has high performance in T-cells and B-cells counting. And our microfluidic cell counting system has great potential as a tool for point-of-care leukocyte analysis system.

The present study was performed to establish a novel ocular surgery simulator for training in peeling of the inner limited membrane (ILM). This simulator included a next-generation artificial ILM with mechanical properties similar to the natural ILM that could be peeled underwater in the same manner as in actual surgery. An artificial eye consisting of a fundus and eyeball parts was fabricated. The artificial eye was installed in the eye surgery simulator. The fundus part was mounted in the eyeball, which consisted of an artificial sclera, retina, and ILM. To measure the thickness of the fabricated ILM on the artificial retina, we calculated the distance of the step height as the thickness of the artificial ILM. Two experienced ophthalmologists then assessed the fabricated ILM by sensory evaluation. The minimum thickness of the artificial ILM was 1.9 ± 0.3 μm (n = 3). We were able to perform the peeling task with the ILM in water. Based on the sensory evaluation, an ILM with a minimum thickness and 1000 degrees of polymerization was suitable for training. We installed the eye model on an ocular surgery simulator, which allowed for the performance of a sequence of operations similar to ILM peeling. In conclusion, we developed a novel ocular surgery simulator for ILM peeling. The artificial ILM was peeled underwater in the same manner as in an actual operation.

The ability to accurately detect and analyze rare cells in a cell population is critical not only for the study of disease progression but also for next flow cytometry systems in clinical application. Here, we report the development of a prototype device, the 'Rare cell sorter', for isolating and recovering single rare cells from whole blood samples. On this device, we utilized an open-channel microfluidic chip for rare cell isolation. And the advantage of open-channel allows us to recover the isolated rare cell directly from the chip. We set the circulating tumor cell (CTC) as a target cell. For the clinical experiment, CTCs were isolated from blood samples collected from patients with metastatic breast cancer and healthy volunteers. There was a significant difference in the number of CTCs between the patients with metastatic breast cancer and healthy volunteers. To evaluate the damage to cells during isolation and recovery, we performed an RNA integrity assay using RNA extracted from CTCs recovered from the chip and found that our process for single CTC isolation and recovery is mild enough for gene analysis of CTCs.

Influenza virus infection can result in changes in the cellular ion levels at 2–3 hours post-infection. More H+ is produced by glycolysis, and the viral M2 proton channel also plays a role in the capture and release of H+ during both viral entry and egress. Then the cells might regulate the intracellular pH by increasing the export of H+ from the intracellular compartment. Increased H+ export could lead indirectly to increased extracellular acidity. To detect changes in extracellular pH of both virus-infected and uninfected cells, pH sensors were synthesized using polystyrene beads (1μm) containing Rhodamine B and Fluorescein isothiocyanate (FITC). The fluorescence intensity of FITC can respond to both pH and temperature. So Rhodamine B was also introduced in the sensor for temperature compensation. Then the pH can be measured after temperature compensation. The sensor was adhered to cell membrane for extracellular pH measurement. The results showed that the multiplication of influenza virus in host cell decreased extracellular pH of the host cell by 0.5–0.6 in 4 hours after the virus bound to the cell membrane, compared to that in uninfected cells. Immunostaining revealed the presence of viral PB1 subunits in the nucleus of virus-bound cells that exhibited extracellular pH changes, but no PB1 subunits are detected in virus-unbound cells where the extracellular pH remained constant.

Muscloskeletal tissues, bone, cartilage, muscles and tendons regulate and support the body's actions and are differentiated from mesenchymal stem cells. These organs generate and/or respond to mechanical stress, which is inevitable in daily life. Among these tissues, cartilages play roles in articular function in joints where shear stress is loaded in combination with cyclical or intermittent compressive force by joint action, and provides a template for bone growth under compressive stress directed along the long axis of long bones. Along with soluble factors, such as cytokines and growth factors, mechanical stress has been recognized as one of the epigenetic factors that regulates the gene expression of various types of cells. Thus, the molecular mechanisms of the mechano-sensing, mechano-transduction, and mechano-response of cells have become a focus of connective tissue biology. Here, we will discuss the mechanisms through which differentiated chondrocytes and mesenchymal cells that are differentiating into chondrocytes respond to various types of mechanical stress by altering their phenotype and how these phenotypic changes are molecularly regulated. We will focus on the roles of cell-extracellular matrix interactions through integrins and downstream signaling pathways involving mitogen-activated protein kinases.

Circulating tumor cells (CTCs) in the blood of patients with epithelial malignancies provide a promising and minimally invasive source for early detection of metastasis, monitoring of therapeutic effects and basic research addressing the mechanism of metastasis. In this study, we developed a new filtration-based, sensitive CTC isolation device. This device consists of a 3-dimensional (3D) palladium (Pd) filter with an 8 µm-sized pore in the lower layer and a 30 µm-sized pocket in the upper layer to trap CTCs on a filter micro-fabricated by precise lithography plus electroforming process. This is a simple pump-less device driven by gravity flow and can enrich CTCs from whole blood within 20 min. After on-device staining of CTCs for 30 min, the filter cassette was removed from the device, fixed in a cassette holder and set up on the upright fluorescence microscope. Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively. Cell spike experiments demonstrated that the recovery rate of tumor cells from blood by this Pd filter device was more than 85%. Single living tumor cells were efficiently isolated from these spiked tumor cells by a micromanipulator, and KRAS mutation, HER2 gene amplification and overexpression, for example, were successfully detected from such isolated single tumor cells. Sequential analysis of blood from mice bearing metastasis revealed that CTC increased with progression of metastasis. Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers. These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.

We developed an active virus filter (AVF) that enables virus enrichment for single virus infection, by using insulator-based dielectrophoresis (iDEP). A 3D-constricted flow channel design enabled the production of an iDEP force in the microfluidic chip. iDEP using a chip with multiple active virus filters (AVFs) was more accurate and faster than using a chip with a single AVF, and improved the efficiency of virus trapping. We utilized maskless photolithography to achieve the precise 3D gray-scale exposure required for fabrication of constricted flow channel. Influenza virus (A PR/8) was enriched by a negative DEP force when sinusoidal wave was applied to the electrodes within an amplitude range of 20 Vp-p and a frequency of 10 MHz. AVF-mediated virus enrichment can be repeated simply by turning the current ON or OFF. Furthermore, the negative AVF can inhibit virus adhesion onto the glass substrate. We then trapped and transported one of the enriched viruses by using optical tweezers. This microfluidic chip facilitated the effective transport of a single virus from AVFs towards the cell-containing chamber without crossing an electrode. We successfully transported the virus to the cell chamber (v = 10 µm/s) and brought it infected with a selected single H292 cell.

Manipulation and injection of single nanosensors with high cell viability is an emerging field in cell analysis. We propose a new method using fluorescence nanosensors with a glass nanoprobe and optical control of the zeta potential. The nanosensor is fabricated by encapsulating a fluorescence polystyrene nanobead into a lipid layer with 1,3,3-trimethylindolino-6′-nitrobenzopyrylospiran (SP), which is a photochromic material. The nanobead contains iron oxide nanoparticles and a temperature-sensitive fluorescent dye, Rhodamine B. The zeta potential of the nanosensor switches between negative and positive by photo-isomerization of SP with ultraviolet irradiation. The positively-charged nanosensor easily adheres to a negatively-charged glass nanoprobe, is transported to a target cell, and then adheres to the negatively-charged cell membrane. The nanosensor is then injected into the cytoplasm by heating with a near-infrared (NIR) laser. As a demonstration, a single 750 nm nanosensor was picked-up using a glass nanoprobe with optical control of the zeta potential. Then, the nanosensor was transported and immobilized onto a target cell membrane. Finally, it was injected into the cytoplasm using a NIR laser. The success rates of pick-up and cell immobilization of the nanosensor were 75% and 64%, respectively. Cell injection and cell survival rates were 80% and 100%, respectively.