Simultaneous Detection and HER2 Profiling of Circulating Breast Cancer Cells in Clinical Patients Using a Rare Cell Sorter.

BACKGROUND/AIM: This study describes a rare cell sorter (RCS) method to detect circulating tumor cells (CTCs) and CTC clusters in whole blood without pretreatment. PATIENTS AND METHODS: We collected samples from breast cancer patients at the University of Tsukuba Hospital. A total of 15 whole-blood specimens from patients with breast cancer were collected and analyzed via a microfluidics chip, fluorescence-conjugated antibody staining, and fluorescence microscopy. Of 15 total cases, eight were analyzed by RCS ver3 and seven were analyzed by RCS ver3.5 to reveal potential clinical differences in scanning methods. We then examined the HER2 status on 4 of the 15 patients using our RCS system. RESULTS: RCS efficiently detected all subtypes of CTCs and CTC clusters from the peripheral blood of cancer patients. The concordance rate of HER2 status between tissue and CTCs in 4 tested clinical samples was 100%. CONCLUSION: RCS is a non-invasive method that allows for simultaneous detection of CTCs, cluster presence, and surface marker (e.g., HER2) status. Frequent sampling is, thus, possible and the large amount of data obtained will be clinically useful to predict response to therapy as well as plan adjunct support therapies in cancer patients.

In vitro magnetosome remineralization for silver-magnetite hybrid magnetosome biosynthesis and used for healing of the infected wound.

BACKGROUND: Magnetosomes (BMPs) are organelles of magnetotactic bacteria (MTB) that are responsible for mineralizing iron to form magnetite. In addition, BMP is an ideal biomaterial that is widely used in bio- and nano-technological applications, such as drug delivery, tumor detection and therapy, and immunodetection. The use of BMPs to create multifunctional nanocomposites would further expand the range of their applications. RESULTS: In this study, we firstly demonstrate that the extracted BMP can remineralize in vitro when it is exposed to AgNO3 solution, the silver ions (Ag+) were transported into the BMP biomembrane (MM) and mineralized into a silver crystal on one crystal plane of Fe3O4. Resulting in the rapid synthesis of an Ag-Fe3O4 hybrid BMP (BMP-Ag). The synergy between the biomembrane, Fe3O4 crystal, and unmineralized iron enabled the remineralization of BMPs at an Ag+ concentration ≥ 1.0 mg mL-1. The BMP-Ag displayed good biocompatibility and antibacterial activity. At a concentration of 2.0 mg/mL, the BMP-Ag and biomembrane removed Ag-Fe3O4 NPs inhibited the growth of gram-negative and gram-positive bacteria. Thus using BMP-Ag as a wound dressing can effectively enhance the contraction of infected wounds. CONCLUSIONS: This study represents the first successful attempt to remineralize organelles ex vivo, realizing the biosynthesis of hybrid BMP and providing an important advancement in the synthesis technology of multifunctional biological nanocomposites.

Bionic eye system mimicking microfluidic structure and intraocular pressure for glaucoma surgery training.

Among increasing eye diseases, glaucoma may hurt the optic nerves and lead to vision loss, the treatment of which is to reduce intraocular pressure (IOP). In this research, we introduce a new concept of the surgery simulator for Minimally Invasive Glaucoma Surgery (MIGS). The concept is comprised of an anterior eye model and a fluidic circulatory system. The model made of flexible material includes a channel like the Schlemm's canal (SC) and a membrane like the trabecular meshwork (TM) covering the SC. The system can monitor IOP in the model by a pressure sensor. In one of the MIGS procedures, the TM is cleaved to reduce the IOP. Using the simulator, ophthalmologists can practice the procedure and measure the IOP. First, considering the characteristics of human eyes, we defined requirements and target performances for the simulator. Next, we designed and manufactured the prototype. Using the prototype, we measured the IOP change before and after cleaving the TM. Finally, we demonstrated the availability by comparing experimental results and target performances. This simulator is also expected to be used for evaluations and developments of new MIGS instruments and ophthalmic surgery robots in addition to the surgical training of ophthalmologists.

Intelligent image-activated cell sorting 2.0.

The advent of intelligent image-activated cell sorting (iIACS) has enabled high-throughput intelligent image-based sorting of single live cells from heterogeneous populations. iIACS is an on-chip microfluidic technology that builds on a seamless integration of a high-throughput fluorescence microscope, cell focuser, cell sorter, and deep neural network on a hybrid software-hardware data management architecture, thereby providing the combined merits of optical microscopy, fluorescence-activated cell sorting (FACS), and deep learning. Here we report an iIACS machine that far surpasses the state-of-the-art iIACS machine in system performance in order to expand the range of applications and discoveries enabled by the technology. Specifically, it provides a high throughput of ∼2000 events per second and a high sensitivity of ∼50 molecules of equivalent soluble fluorophores (MESFs), both of which are 20 times superior to those achieved in previous reports. This is made possible by employing (i) an image-sensor-based optomechanical flow imaging method known as virtual-freezing fluorescence imaging and (ii) a real-time intelligent image processor on an 8-PC server equipped with 8 multi-core CPUs and GPUs for intelligent decision-making, in order to significantly boost the imaging performance and computational power of the iIACS machine. We characterize the iIACS machine with fluorescent particles and various cell types and show that the performance of the iIACS machine is close to its achievable design specification. Equipped with the improved capabilities, this new generation of the iIACS technology holds promise for diverse applications in immunology, microbiology, stem cell biology, cancer biology, pathology, and synthetic biology.

Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering.

Combining the strength of flow cytometry with fluorescence imaging and digital image analysis, imaging flow cytometry is a powerful tool in diverse fields including cancer biology, immunology, drug discovery, microbiology, and metabolic engineering. It enables measurements and statistical analyses of chemical, structural, and morphological phenotypes of numerous living cells to provide systematic insights into biological processes. However, its utility is constrained by its requirement of fluorescent labeling for phenotyping. Here we present label-free chemical imaging flow cytometry to overcome the issue. It builds on a pulse pair-resolved wavelength-switchable Stokes laser for the fastest-to-date multicolor stimulated Raman scattering (SRS) microscopy of fast-flowing cells on a 3D acoustic focusing microfluidic chip, enabling an unprecedented throughput of up to ∼140 cells/s. To show its broad utility, we use the SRS imaging flow cytometry with the aid of deep learning to study the metabolic heterogeneity of microalgal cells and perform marker-free cancer detection in blood.

Development of a Spherical Model with a 3D Microchannel: An Application to Glaucoma Surgery.

Three-dimensional (3D) microfluidic channels, which simulate human tissues such as blood vessels, are useful in surgical simulator models for evaluating surgical devices and training novice surgeons. However, animal models and current artificial models do not sufficiently mimic the anatomical and mechanical properties of human tissues. Therefore, we established a novel fabrication method to fabricate an eye model for use as a surgical simulator. For the glaucoma surgery task, the eye model consists of a sclera with a clear cornea; a 3D microchannel with a width of 200-500 µm, representing the Schlemm's canal (SC); and a thin membrane with a thickness of 40-132 µm, representing the trabecular meshwork (TM). The sclera model with a clear cornea and SC was fabricated by 3D molding. Blow molding was used to fabricate the TM to cover the inner surface of the sclera part. Soft materials with controllable mechanical behaviors were used to fabricate the sclera and TM parts to mimic the mechanical properties of human tissues. Additionally, to simulate the surgery with constraints similar to those in a real operation, the eye model was installed on a skull platform. Therefore, in this paper, we propose an integration method for fabricating an eye model that has a 3D microchannel representing the SC and a membrane representing the TM, to develop a glaucoma model for training novice surgeons.

Fabrication of 3D Capillary Vessel Models with Circulatory Connection Ports.

Bionic microscopic vessel models can contribute to the development of vascular treatment skills and techniques for clinical training. Most microscopic vessel models are limited to two dimensions, but three-dimensional (3D) models are important for surgery, such as on retina microscopic vessels, for the observation of colon microvessels, for measuring the deformability of red blood cell (RBC), and so on. Therefore, bionic 3D blood vessel models are increasingly in demand. For this reason, it is necessary to establish 3D fabrication techniques for microchannels. In this study, we established two fabrication methods for 3D microfluidic devices for the development of microscopic vessel models. First, we employed an exposure method using photolithographic technology. Second, we employed a 3D method using femtosecond laser and mask hybrid exposure (FMEx). Both methods made it possible to fabricate a millimeter-scale 3D structure with a submicrometer resolution and achieve an easy injection of solution. This is because it was possible to fabricate typical microfluidic channels used for model inlet and outlet ports. Furthermore, in the FMEx method, we employed an acid-diffusion effect using a chemically amplified resist to form a circular channel cross-section. The acid-diffusion effect made it realizable to fabricate a smooth surface independent of the laser scanning line width. Thus, we succeeded in establishing two methods for the fabrication of bionic 3D microfluidic devices with microfluidic channels having diameters of 15⁻16 µm for mimicking capillary vessels.

Autonomous Positioning of Eye Surgical Robot Using the Tool Shadow and Kalman Filtering.

Vitreoretinal surgery is one of the most difficult surgical operations, even for experienced surgeons. Thus, a master-slave eye surgical robot has been developed to assist the surgeon in safely performing vitreoretinal surgeries; however, in the master-slave control, the robotic positioning accuracy depends on the surgeon's coordination skills. This paper proposes a new method of autonomous robotic positioning using the shadow of the surgical instrument. First, the microscope image is segmented into three regions-namely, a micropipette, its shadow, and the eye ground-using a Gaussian mixture model (GMM). The tips of the micropipette and its shadow are then extracted from the contour lines of the segmented regions. The micropipette is then autonomously moved down to the simulated eye ground until the distance between the tips of micropipette and its shadow in the microscopic image reaches a predefined threshold. To handle possible occlusions, the tip of the shadow is estimated using a Kalman filter. Experiments to evaluate the robotic positioning accuracy in the vertical direction were performed. The results show that the autonomous positioning using the Kalman filter enhanced the accuracy of robotic positioning.

Influenza virus replication raises the temperature of cells.

Influenza virus invades the cell by binding sialic acid on the cell membrane through haemagglutinin (HA), and then genome replication and transcription are carried out in the nucleus to produce progeny virus. Multiplication of influenza virus requires metabolites, such as nucleotides and amino acids, as well as cellular machinery to synthesize its genome and proteins, thereby producing viral particles. Influenza virus infection forces the start of several metabolic systems in the cell, which consume or generate large amounts of energy. Thus, the viral multiplication processes involved in both genome replication and transcription are considered to require large numbers of nucleotides. The high-level consumption of nucleotides generates large amounts of energy, some of which is converted into heat, and this heat may increase the temperature of cells. To address this question, we prepared a tool based on rhodamine B fluorescence, which we used to measure the temperatures of influenza virus-infected and uninfected cells. The results indicated that influenza virus multiplication increased the temperature of cells by approximately 4 °C - 5 °C, ATP levels in the cells decreased at 3 h after infection, and mitochondrial membrane potential decreased with multiplication level. Thus, the increase in cellular temperature during influenza virus infection appears to be due to the massive consumption of ATP over a short period.

A surgical simulator for peeling the inner limiting membrane during wet conditions.

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.

Three-Dimensional Blood Vessel Model with Temperature-Indicating Function for Evaluation of Thermal Damage during Surgery.

Surgical simulators have recently attracted attention because they enable the evaluation of the surgical skills of medical doctors and the performance of medical devices. However, thermal damage to the human body during surgery is difficult to evaluate using conventional surgical simulators. In this study, we propose a functional surgical model with a temperature-indicating function for the evaluation of thermal damage during surgery. The simulator is made of a composite material of polydimethylsiloxane and a thermochromic dye, which produces an irreversible color change as the temperature increases. Using this material, we fabricated a three-dimensional blood vessel model using the lost-wax process. We succeeded in fabricating a renal vessel model for simulation of catheter ablation. Increases in the temperature of the materials can be measured by image analysis of their color change. The maximum measurement error of the temperature was approximately -1.6 °C/+2.4 °C within the range of 60 °C to 100 °C.

On-chip cell sorting by high-speed local-flow control using dual membrane pumps.

Although researchers have proposed various methods of on-chip cell sorting, high-throughput sorting of large cells remains hampered by the difficulty of controlling high-speed flow over a wide sorting area. To overcome this problem, we proposed high-speed local-flow control using dual membrane pumps driven by piezoelectric actuators placed on the outside of a microfluidic chip in this paper. We evaluated the controllability of shifting the flow profile by the local-flow. The results indicated that we could sort large cells up to approximately 150 µm in size with an equivalent throughput of 31 kHz. Because our method can control the flow profiles, it is applicable not only to large cells but also to small cells. The cell-sorting efficacy of the proposed method was experimentally evaluated on Euglena gracilis NIES-48 (E. gracilis) cells as large target cells and GCIY-EGFP (GCIY) cells derived from a gastric cancer cell line as small target cells. In E. gracilis cells sorting, the throughput is 23 kHz with a 92.8% success rate, 95.8% purity, and 90.8% cell viability. In GCIY sorting, the throughput is 11 kHz with a 97.8% success rate, 98.9% purity, and 90.7% cell viability. These results confirm that the proposed method sorts differently sized cells with high throughput and hence, overcomes the throughput-size trade-off that exists in conventional on-chip cell sorters.

Rare cell isolation and recovery on open-channel microfluidic chip.

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.

Mechanical diagnosis of human erythrocytes by ultra-high speed manipulation unraveled critical time window for global cytoskeletal remodeling.

Large deformability of erythrocytes in microvasculature is a prerequisite to realize smooth circulation. We develop a novel tool for the three-step "Catch-Load-Launch" manipulation of a human erythrocyte based on an ultra-high speed position control by a microfluidic "robotic pump". Quantification of the erythrocyte shape recovery as a function of loading time uncovered the critical time window for the transition between fast and slow recoveries. The comparison with erythrocytes under depletion of adenosine triphosphate revealed that the cytoskeletal remodeling over a whole cell occurs in 3 orders of magnitude longer timescale than the local dissociation-reassociation of a single spectrin node. Finally, we modeled septic conditions by incubating erythrocytes with endotoxin, and found that the exposure to endotoxin results in a significant delay in the characteristic transition time for cytoskeletal remodeling. The high speed manipulation of erythrocytes with a robotic pump technique allows for high throughput mechanical diagnosis of blood-related diseases.

Real-time in vitro intravascular reconstruction and navigation for endovascular aortic stent grafting.

BACKGROUND: Trans-catheter endovascular stent grafting minimizes trauma and increases the benefitting patient population. However, the alignment between stent graft branches and vasculature branches remains time-consuming and challenging, and such techniques require a significant amount of contrast agent for imaging. METHODS: A new framework for intravascular reconstruction based on sensor fusion between intravascular ultrasound (IVUS) imaging and electromagnetic (EM) tracking was proposed. A new image processing method was presented to realize fully automatic processing of IVUS imaging and 3D reconstruction in real time, as well as branch detection for alignment and deployment. Complementary navigation using CT data allows for efficient catheter advancement and assistant clinical judgement. RESULTS: The reconstruction of an in vitro descending aorta phantom with branches was realized at 35 Hz, with cross-section radius average error of 0.64 mm. CONCLUSION: The proposed method demonstrates significant potential for clinical applications, enables navigation for precise alignment and placement for stent grafting to reduce surgical time, and decreases hemorrhagic collisions and the use of contrast agent. Copyright © 2016 John Wiley & Sons, Ltd.

Egg-in-cube: design and fabrication of a novel artificial eggshell with functionalized surface.

An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed "Egg-in-Cube" with functionalized surface has great potential to serve as a promising platform for biomedical research.

Quantitative study on appearance of microvessels in spectral endoscopic imaging.

Increase in abnormal microvessels in the superficial mucosa is often relevant to diagnostic findings of neoplasia in digestive endoscopy; hence, observation of superficial vasculature is crucial for cancer diagnosis. To enhance the appearance of such vessels, several spectral endoscopic imaging techniques have been developed, such as narrow-band imaging and blue laser imaging. Both techniques exploit narrow-band blue light for the enhancement. The emergence of such spectral imaging techniques has increased the importance of understanding the relation of the light wavelength to the appearance of superficial vasculature, and thus a new method is desired for quantitative analysis of vessel visibility in relation to the actual structure in the tissue. Here, we developed microvessel-simulating phantoms that allowed quantitative evaluation of the appearance of 15-µm-thick vessels. We investigated the relation between the vascular contrast and light wavelength by the phantom measurements and also verified it in experiments with swine, where the endoscopically observed vascular contrast was investigated together with its real vascular depth and diameter obtained by microscopic observation of fluorescence-labeled vessels. Our study indicates that changing the spectral property even in the wavelength range of blue light may allow selective enhancement of the vascular depth for clinical use.

Quantitative assessment of manual and robotic microcannulation for eye surgery using new eye model.

BACKGROUND: Microcannulation, a surgical procedure for the eye that requires drug injection into a 60-90 µm retinal vein, is difficult to perform manually. Robotic assistance has been proposed; however, its effectiveness in comparison to manual operation has not been quantified. METHODS: An eye model has been developed to quantify the performance of manual and robotic microcannulation. The eye model, which is implemented with a force sensor and microchannels, also simulates the mechanical constraints of the instrument's movement. Ten subjects performed microcannulation using the model, with and without robotic assistance. RESULTS: The results showed that the robotic assistance was useful for motion stability when the drug was injected, whereas its positioning accuracy offered no advantage. CONCLUSIONS: An eye model was used to quantitatively assess the robotic microcannulation performance in comparison to manual operation. This approach could be valid for a better evaluation of surgical robotic assistance.

Development of a new rapid isolation device for circulating tumor cells (CTCs) using 3D palladium filter and its application for genetic analysis.

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.

In vitro simulator with numerical stress analysis for evaluation of stent-assisted coiling embolization in cerebral aneurysm treatments.

BACKGROUND: There are several complications associated with Stent-assisted Coil Embolization (SACE) in cerebral aneurysm treatments, due to damaging operations by surgeons and undesirable mechanical properties of stents. Therefore, it is necessary to develop an in vitro simulator that provides both training and research for evaluating the mechanical properties of stents. METHODS: A new in vitro simulator for three-dimensional digital subtraction angiography was constructed, followed by aneurysm models fabricated with new materials. Next, this platform was used to provide training and to conduct photoelastic stress analysis to evaluate the SACE technique. RESULTS: The average interaction stress increasingly varied for the two different stents. Improvements for the Maximum-Likelihood Expectation-Maximization method were developed to reconstruct cross-sections with both thickness and stress information. CONCLUSIONS: The technique presented can improve a surgeon's skills and quantify the performance of stents to improve mechanical design and classification. This method can contribute to three-dimensional stress and volume variation evaluation and assess a surgeon's skills.